Genetically modified fungi and their use in lipid production

ABSTRACT

The invention refers to fungal cells, and especially to oleaginous fungal cells that have been genetically modified to produce enzymes of the pyruvate dehydrogenase bypass route to enhance their lipid production. Especially the cells are modified to overexpress genes encoding pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and/or acetyl-CoA synthetase (ACS), optionally together with a gene encoding diacylglycerol acyltransferase (DAT), or to express genes encoding PDC together with ALD and/or ACS. Methods of producing lipids, biofuels and lubricants using the modified fungi are also disclosed as well as expression cassettes useful therein. A new enzyme having phosholipid:diacylglycerol acyltransferase (PDAT) activity and a polynucleotide encoding it are also disclosed, which are useful in the lipid production. A recombinant  Cryptococcus  cell and its construction is described.

This application is a Divisional of copending application Ser. No.13/806,281, filed on Dec. 21, 2012, which was filed as PCT InternationalApplication No. PCT/FI2011/050594 on Jun. 21, 2011, which claims thebenefit under 35 U.S.C. §119(a) to Patent Application No. 20105733,filed in FINLAND on Jun. 24, 2010, all of which are hereby expresslyincorporated by reference into the present application.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“0837_0322PUS2_SequenceListing.txt” created on Dec. 21, 2012 and is58,075 bytes in size. The sequence listing contained in this .txt fileis part of the specification and is hereby incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to fungi, which have been geneticallymodified to enhance their lipid production. The invention also relatesto a method for preparing the fungi, and to expression cassettes forgenetic modification of the fungi. The invention further relates to amethod of producing lipids by these fungi. The lipids produced areuseful in manufacturing biofuels, lubricants and functional fatty acids.The invention thus also provides a method for producing biofuels andlubricants. Still further the invention provides a new enzyme proteinthat is useful in the methods, and a nucleic acid encoding it. Evenfurther the invention relates to a recombinant Cryptococcus cell, and amethod for its construction.

BACKGROUND OF THE INVENTION

Biofuels are current favorites to be the next generation transportationfuels. They are produced from renewable biological sources such asvegetable oils and animal fats. They are biodegradable, non-toxic andhave a low emission profile. Due to the limited sources of biodiesel rawmaterials such as rape seed oil, soy bean oil or palm oil, it is ofimportance to expand biodiesel raw materials to non-food materials likemicrobes. The benefits of using microbes for production of oils are:they are affected neither by seasons nor by climates, they are able toproduce high lipid contents, and the oils can be produced from a widevariety of sources with short production times, especially from residueswith abundant nutrition. Microbiologically produced lipids may also beused e.g. for the production of functional fatty acids.

A few fungal species accumulate remarkable amounts of lipid in thecells. It has been observed that lipids accumulate in these so calledoleaginous fungi under nitrogen limited conditions, which has resultedin a hypothesis for effective lipid accumulation (Review Ratledge andWynn 2002 and references thenceforth). Nitrogen limitation causesactivation of the AMP deaminase which utilizes AMP to produce NH₄. Thedecrease in AMP concentration inhibits the activity of mitochondrialisocitrate dehydrogenase (IDH) which is part of the mitochondrialtricarboxylic (TCA) cycle. Reduction in IDH activity results inequilibration of isocitrate to citrate by aconitase. Produced citrate istransferred to the cytosol where it is converted with Coenzyme A (CoA)to acetyl-CoA by ATP:citrate lyase (ACL) with ATP hydrolysis.

Cytosolic acetyl-CoA can be further used in fatty acid synthesis. Acomprehensive review on fatty acid synthesis and elongation in yeast,especially in Saccharomyces cerevisiae, is that of Tehlivets et al.,1997. In the first step of fatty acid synthesis acetyl-CoA iscarboxylated by the addition of carbon dioxide to malonyl-CoA by theenzyme acetyl-CoA carboxylase in an ATP demanding reaction. In thefollowing reactions by the fatty acid synthase systems acyl and malonylmoieties from acyl-CoA and malonyl-CoA, respectively, are transferred toacyl carrier proteins (ACPs), after the acyl chain, typically initiatedby acetyl-CoA, is condensated with malonyl-ACP followed by reduction ofthe 3-ketoacyl-ACP to 3-hydroxyacyl-ACP, dehydration to enoyl-ACP, and asecond reduction to a saturated acyl-chain that is extended by twocarbon atoms. These synthesis steps are usually repeated seven timesresulting in palmitoyl ACP (C16:0). Palmitic acid and intermediates ofthe fatty acid synthesis after hydrolysed to acyl-CoAs byhydrolase/thioesterase, can be further modified by different elongasesand desaturases to different length acyl-chains with or without doublebonds. In one cycle of fatty acid synthesis two NADPHs are required inthe reduction steps. Acyl-CoAs can be further synthesised totriacylglycerols.

Triacylglycerol synthesis starts from glycerol-3-phosphate ordihydroxyacetone phosphate which is acylated (dihydroxyacetone-phosphatealso reduced) to 1-acyl-glycerol-3-phosphate which is further acylatedto phosphatidic acid. Phosphatidic acid can be further dephosphorylatedto diacylglycerol. Diacylglycerol is further acylated to triacylglycerolmainly by acyl-CoA:diacylglycerol acyltransferase (DGAT) andphospholipid:diacylglycerol acyltransferase (PDAT) utilizing acyl-CoA orphosphatidylcholine, respectively, as acyl donors. The triacylglycerolpathway in yeast S. cerevisiae is described in more detail in amini-review of Sorger and Daum 2003.

Phospholipid:diacylglycerol acyltransferase (PDAT) encoding genesoriginating from S. cerevisiae and Yarrowia lipolytica have beenexpressed in yeasts S cerevisiae and Y. lipolytica to enhance theirtriacylglycerol production (WO00/60095 and WO2005/003322, respectively).WO2009/126890 provides systems for producing engineered oleaginous yeastor fungi that express caroteinoids. Oleaginy is promoted e.g. byincreased or heterologous expression of DGAT or PDAT, whereas reducingthe activity of PDC is expected to promote oleaginy.

Methods of manufacturing biodiesel and other oil-based compounds usingglycerol as an energy source in fermentation of oil-bearingmicroorganisms have been described e.g. in US2009/0004715. Methods ofproducing lipid-based biofuels from cellulose containing feedstock byheterotrophic fermentation of microorganisms have been described inUS2009/0064567. Both publications focus on the use of algae as lipidproducers. No details are given. WO2007/136762 provides geneticallyengineered microorganisms that produce desired products from the fattyacid biosynthetic pathway.

With the above-described triacylglycerol production pathway hightriglyceride yields indicated as triglyceride production per used carbonsource cannot be achieved or triacylglycerol production per cell biomasscannot be significantly enhanced. In general, lipids especiallytriglycerides are produced when nitrogen becomes a growth limitingfactor at the late logarithmic or early stationary growth phaseresulting in a low triglyceride production rate compared e.g. to yeastethanol production. Additionally, the need of several carbons andreduced cofactors in synthesis of triacylglycerol result in low yieldper used carbon. The present invention uses another route for microbiallipid production. In the present invention microbial lipid productionrate and yields are enhanced, and the need of reduced cofactors from theoutside of the lipid pathway is decreased. The present invention furtherprovides lipid production that is not linked to nitrogen limitation.

SUMMARY OF THE INVENTION

The present invention is based on the use of a pyruvate dehydrogenasebypass route for producing cytosolic acetyl-CoA. The invention makes useof an active cytosolic pathway for acetyl-CoA production, which proceedsvia the enzymatic reaction catalyzed by pyruvate decarboxylase (PDC).This pathway is known in Crabtree-positive yeast S. cerevisiae, where itis essential for cytosolic acetyl-CoA production, but it has not beencharacterized in oleaginous yeasts and oleaginous filamentous fungi. Inoleaginous fungi another cytosolic pathway for acetyl-CoA production,proceeding via the reaction catalyzed by pyruvate dehydrogenase (PDH) iswell known and which has been shown to be essential for cytosolicacetyl-CoA production from pyruvate. This pathway operates viamitochondria and in this pathway a higher fraction of carbon is lostthan in the pathway via pyruvate decarboxylase. In the pyruvatedecarboxylase pathway, exploited in this invention, a higher fraction ofcarbon is directed to triacylglycerol and it is not dependent onmitochondrial enzyme activities. Further, it produces NAD(P)H which isneeded in the following fatty acid synthesis. The pyruvate dehydrogenasebypass route optionally together with an enhanced diacylglycerolacyltransferase activity as provided by the invention removes manybottlenecks in microbial lipid production.

The invention is directed to genetically modified fungal cells that havebeen modified to enhance the expression of a nucleic acid encoding PDC,ALD, ACS and/or DAT, and to methods of constructing them.

In particular the invention is directed to a genetically modifiedoleaginous fungal cell comprising at least one nucleic acid withenhanced expression encoding an enzyme selected from the groupconsisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase(ALD) and acetyl-CoA synthetase (ACS).

The invention is further directed to a genetically modified fungal cellcomprising:

a) a nucleic acid with modified expression encoding a pyruvatedecarboxylase (PDC) enzyme, and

b) at least one nucleic acid with modified expression encoding an enzymeselected from the group consisting of acetaldehyde dehydrogenase (ALD),acetyl-CoA synthetase (ACS) and diacylglycerol acyltransferase (DAT).

The invention is also directed to a genetically modified fungal cellcomprising:

a) at least one nucleic acid with modified expression encoding an enzymeselected from the group consisting of pyruvate decarboxylase (PDC),acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), and

b) a nucleic acid with modified expression encoding a diacylglycerolacyltransferase (DAT) enzyme.

The invention is also directed to a method of producing lipidscomprising cultivating the genetically modified fungal cell according tothe invention in a medium containing carbon and nitrogen sources, andrecovering the lipids produced.

The invention is further directed to a method of producing biofuel, orlubricant said method comprising cultivating the genetically modifiedfungal cell according to the invention in a medium containing carbon andnitrogen sources, and recovering the lipids produced, and optionallyesterifying said lipids to obtain biodiesel or lubricant, orhydrogenizing the lipids to obtain renewable diesel or lubricant.

The invention is still further directed to methods of preparing i.e.constructing a genetically modified fungal cell of the invention, saidmethods comprising transforming a fungal cell with

at least one nucleic acid with enhanced expression encoding an enzymeselected from the group consisting of pyruvate decarboxylase (PDC),acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS); orwith

a) a nucleic acid with enhanced expression encoding a pyruvatedecarboxylase (PDC) enzyme, and

b) at least one nucleic acid with enhanced expression encoding an enzymeselected from the group consisting of acetaldehyde dehydrogenase (ALD),acetyl-CoA synthetase (ACS) and diacylglycerol acyltransferase (DAT); orwith

a) at least one nucleic acid with modified expression encoding an enzymeselected from the group consisting of pyruvate decarboxylase (PDC),acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), and

b) a nucleic acid with modified expression encoding a diacylglycerolacyltransferase (DAT) enzyme.

In addition the invention is directed to an expression cassettecomprising

a) at least one nucleic acid with modified expression encoding an enzymeselected from the group consisting of pyruvate decarboxylase (PDC),acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), and

b) a nucleic acid with modified expression encoding a diacylglycerolacyltransferase (DAT) enzyme.

Conveniently the genetically modified fungal cells are constructedaccording to the invention by transforming the fungal cell with thenucleic acid(s) encoding said enzyme(s) resulting in enhanced enzymeactivity of said enzyme.

Still further the invention is directed to an enzyme protein havingphosholipid:diacylglycerol acyltransferase (PDAT) activity and at least40% sequence identity to SEQ ID NO:52, or an enzymatically activefragment or variant thereof, and to an isolated nucleic acid moleculeselected from the group consisting of: a) a nucleic acid encoding saidprotein, b) a nucleic acid comprising the nucleotide sequence of SEQ IDNO:53 or SEQ ID NO:93, c) a complementary strand of a) or b), and d) asequence that is degenerate as a result of the genetic code to anyone ofsequences a)-c).

The invention is additionally directed to a genetically modified fungalcell comprising a nucleic acid with modified expression encoding saidPDAT, and to a method of producing lipids, comprising cultivating saidgenetically modified fungal cell in a medium containing carbon andnitrogen sources, and recovering the lipids produced.

Still further the invention is directed to the use of a geneticallymodified fungal cell of the invention for producing lipids, biofuels,biodiesel, renewable diesel or lubricants. The use for producing lipidsincludes e.g. the use for producing precursors of fatty acids e.g. offunctional fatty acids, and for producing the fatty acids or functionalfatty acids.

As a still further aspect, the invention is directed to a geneticallymodified Cryptococcus cell, which has been modified to enhance theexpression of a heterologous nucleic acid, and to a method ofconstructing the cell, said method comprising transforming aCryptococcus cell with a nucleic acid encoding a heterologous protein.

Specific embodiments of the invention are set forth in the dependentclaims. Other objects, details and advantages of the present inventionwill become apparent from the following drawings, detailed descriptionand examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows probable metabolic routes for cytosolic acetyl-CoAproduction via the pyruvate dehydrogenase route in grey, and via thepyruvate dehydrogenase bypass route i.e. the pyruvate decarboxylaseroute in black.

FIG. 2 shows the metabolic route for triacylglycerol production.

FIG. 3 is a diagram depicting plasmid pKK81.

FIG. 4 is a diagram depicting plasmid pKK82.

FIG. 5 is a diagram depicting plasmid pKK86.

FIG. 6 is a diagram depicting plasmid pKK95.

FIG. 7 is a diagram depicting plasmid pKK85.

FIG. 8 is a diagram depicting plasmid pKK75.

FIG. 9 is a diagram depicting plasmid pKK94.

FIG. 10 is a diagram depicting plasmid pKK96.

FIG. 11 is a diagram depicting plasmid pKK98.

FIG. 12 is a diagram depicting plasmid pKK101.

FIG. 13 is a diagram depicting plasmid pKK102.

DETAILED DESCRIPTION OF THE INVENTION

The presumed pyruvate dehydrogenase (PDH) pathway for producingcytosolic acetyl-CoA is shown in grey in FIG. 1. First cytosolicpyruvate is transported into the mitochondria where it is oxidativelydecarboxylated to acetyl-CoA and carbon dioxide by the pyruvatedehydrogenase complex. The resulting acetyl-CoA is then entering thetricarboxylic (TCA) cycle by citrate synthase (CS) which catalyses thecondensation reaction of the two-carbon acetate residue from acetyl-CoAand a molecule of four-carbon oxaloacetate (OAA) to form the six-carboncitrate. Citrate is then isomerised to isocitrate which is the substratefor mitochondrial isocitrate dehydrogenase (IDH). Under limited nitrogensupply AMP deaminase activity increases leading to production of IMP andammonium from AMP. The decrease in the amount of AMP results in decreasein mitochondrial isocitrate dehydrogenase (IDH) activity, whereby theamount of citrate in the mitochondria increases. The mitochondrialcitrate is transported into the cytosol, where ATP:citrate lyase (ACL)converts citrate, ATP and CoA into acetyl-CoA and oxaloacetate. Theoxaloacetate is degraded by malate dehydrogenase (MDH) to malate, whichin turn is converted to pyruvate and carbon dioxide by malic enzyme(MAE) under production of NADPH, which is an important cofactor in fattyacid synthesis. In this invention cytosolic acetyl-CoA is produced via apyruvate dehydrogenase bypass pathway, which is shown in black inFIG. 1. This pathway is also called the pyruvate decarboxylase pathway.In this pathway pyruvate is decarboxylated to acetaldehyde and carbondioxide by pyruvate decarboxylase (PDC). Acetaldehyde is furtheroxidised to acetate by NADP⁺ (or NAD⁺)-dependent acetaldehydedehydrogenase (ALD). Acetate is then converted to acetyl-CoA byacetyl-CoA synthetase (ACS) with ATP and Coenzyme A. In this cytosolicpathway [pyruvate+CoA+ATP+NAD(P)⁺=Acetyl-CoA+CO₂+NAD(P)H+AMP+PPi+H⁺] theoverall acetyl-CoA yield is higher than in the pyruvatedehydrogenase-ATP:citrate lyase pathway [pyruvate+CoA+ATP+NAD⁺ (inmitochondria)+oxaloacetate (in mitochondria)=Acetyl-CoA+CO₂+ADP+Pi+NAD⁺(in mitochondria)+H⁺+oxaloacetate (in cytosol)].

Cytosolic acetyl-CoA is used by NADPH-dependent fatty acid synthase(FAS) and other enzymes for the production of acyl-CoA esters ofdifferent length, which then are attached to for example glycerolthrough the triglyceride metabolic pathway shown in FIG. 2. In the laststep of this pathway an acyl group from acyl-CoA or from a phospholipidis attached to the diacylglycerol by acyl-CoA:diacylglycerolacyltransferase (DGAT) or phospholipid:diacylglycerol acyltransferase(PDAT).

In the present invention triacylglycerol production of fungi will beenhanced by overexpressing at least one gene, which encodes an enzymeinvolved in the pyruvate decarboxylate pathway for converting pyruvateto acetyl-CoA as shown in FIG. 1, together with a gene, which encodes anenzyme that catalyses the acylation of diacylglycerol to triacylglycerolas shown in FIG. 2.

Lipid production including triacylglycerol production in the cell is ahighly NADPH demanding process. E.g. in production of 1 mole of oleicacid (9-octadecenoic acid) 17 mole of NADPH is needed. NADPH produced bymalic enzyme has been proposed to be the main source for NADPH needed infatty acid synthesis. Said NADPH production occurs totally outside thecytosolic acetyl-CoA production pathway resulting in the consumption ofextra carbons in NADPH production, even though the reaction of the malicenzyme is linked to the degradation of the oxaloacetate produced byATP:citrate lyase. In the present invention fatty acid and furthertriacylglycerol production is connected directly to NADPH cofactorproduction by NADP-dependent acetaldehyde dehydrogenase thus reducingthe need to produce NADPH outside the triglyceride production pathwayresulting in an increased triacylglycerol yield. The NADP⁺-dependentacetaldehyde dehydrogenase produces simultaneously one NADPH and oneacetate molecule from NADP+ and acetaldehyde resulting in production ofone mole of NADPH per one mole of pyruvate. This means that half of theNADPH molecules needed in the fatty acid synthesis are producedsimultaneously with the cytosolic acetyl-CoA production. Thissimultaneous NADPH production with lower carbon loss during productionof cytosolic acetyl-CoA results in a better yield in fatty acidproduction following also better yield in triacylglycerol production. Inthis invention only one carbon is lost from the carbon skeletondownstream of pyruvate prior to cytosolic acetyl-CoA. Additionally, noside reactions are needed to cleave metabolites further outside thetriglyceride production pathway. The production of cytosolic acetyl-CoAvia the pyruvate dehydrogenase bypass completes the existing pyruvatedehydrogenase pathway for cytosolic acetyl-CoA production.

Triacylglycerols and other lipids are naturally produced in fungi viathe pyruvate dehydrogenase pathway during growth, but the maintriacylglycerol and lipid accumulation occurs when excess citrate willbe available after nitrogen limitation in the late stage of cultivation.This triacylglycerol production in late logarithmic or stationary phasesof cultivation results in low triacylglycerol production rates,especially at the early stage of cultivation. In this inventionexpression of the pyruvate dehydrogenase bypass catalyzed by PDC, ALD,and ACS results in a situation where triacylglycerol accumulation is notlinked to nitrogen limitation thus allowing enhanced triacylglycerolproduction during cultivation resulting in a better triacylglycerolproduction rate. The earlier triacylglycerol production is furtherenhanced by expressing an acyltransferase such as aphospholipid:diacylglycerol acyltransferase (PDAT) encoding gene e.g.under a constitutive promoter thus increasing triacylglycerolconcentration at the expense of phospholipids.

Contrary to oleaginous yeasts and moulds like Cryptococcus curvatus andMucor circinelloides, S. cerevisiae lacks the pyruvate dehydrogenaseroute for acetyl-CoA production. In this Crabtree-positive yeastcytosolic acetyl-CoA, and further fatty acids and triacylglycerols, areproduced only via pyruvate dehydrogenase bypass. The essential role ofthe pyruvate dehydrogenase bypass including the enzymes pyruvatedecarboxylate (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoAsynthetase (ACS) in cytosolic acetyl-CoA and further in lipid productionand the role of ALD in the generation of reducing equivalents (NADH andNADPH) in S. cerevisiae has been described for example in Flikweert etal. 1996, Pronk et al 1996, Saint-Prix et al 2004). In US2009/0053797expression of endogenous NADP-dependent acetaldehyde dehydrogenase(ALD6) gene and native or modified endogenous ACS1 gene or Salmonellaenterica acetyl-CoA synthetase (ACS1) gene in S. cerevisiae resulted inan increased concentration of cytosolic acetyl-CoA in the production ofisoprenoids. Shiba et al., 2007 found that overexpression of ALD6 andACS1 in S. cerevisiae increased cytosolic acetyl-CoA derivedamorphadiene overproduction, whereas overexpression of ACS2 with ALD6did not. The acetyl-CoA synthetase isoforms ACS1 and ACS2 behavedifferently in S. cerevisiae: ACS1 gene has been shown to be underglucose repression whereas ACS2 gene has been shown to be constitutivelyexpressed and co-regulated with structural genes of fatty acidbiosynthesis (van den Berg et al 1996, Hiesinger et al. 1997).

The essential role of the PDH bypass in S. cerevisiae has beendescribed: Deletion of three structural genes for pyruvate decarboxylase(PDC1, PDC5 and PDC6) results in loss of growth in a defined glucosemedium (Flikweert et al 1996, Pronk et al. 1996), and deletion of twoacetyl-CoA synthetase encoding genes ACS1 and ACS2 is lethal on allcarbon sources (Takahashi et at 2006 and van den Berg et at 1996),indicating that there is no alternative route for cytosolic acetyl-CoAproduction in S. cerevisiae. The enhanced expression of this pathway hasbeen found to induce cytosolic acetyl-CoA production in S. cerevisiae(e.g. US2009/0053797 and WO2008/080124). However, the existence andfunctionality of the pyruvate dehydrogenase bypass pathway in oleaginousyeasts or moulds has not been described in the literature.

PDC has been characterised e.g. from the Rhizopus oryzae, which inaddition to lipids produced ethanol (Skory 2003). Also, ALD and ACS havebeen characterised from some of the oleaginous fungi e.g. A. nidulans(Flipphi et al. 2001, Connerton et al. 1990). Still, it has also beenshown that deletion of the only cytoplasmic ACS encoding gene from A.nidulans had no effect on growth on glucose (Sandeman et al. 1989).Instead, it has been shown in several articles that the cytosolicacetyl-CoA for lipid production will be produced via pyruvatedehydrogenase and ATP:citrate lyase (ACL) in oleaginous fungi (Wynn etal 2001, Boulton and Ratledge 1981). ATP:citrate lyase has been shown tobe absent from the non-oleaginous yeasts (Boulton and Ratledge 1981).E.g. ACL is absent from the sequenced members of the Saccharomycotinawith the exception of Y. lipolytica which is oleaginous yeast. Theessential role of ACL for cytosolic acetyl-CoA production has also beenshown at a functional level by deteting the acl gene from A. nidulans.This deletion strain could not grow in the absence of external sourcesof cytoplasmid acetyl-CoA, which strongly suggests that ACL activity isrequired to generate cytoplasmic acetyl-CoA. This also indicates theabsence of any pyruvate dehydrogenase bypass pathway, which couldcompensate acl deletion (Hynes and Murray 2010). The production ofcytosolic acetyl-CoA via ATP:citrate lyase in oleaginous fungi is alsosuggested in the patent applications WO2005/003322 and US2006/094087,where diacylglycerol transferase encoding genes have been expressed toenhance triacylglycerol production.

The term “pyruvate dehydrogenase bypass” refers to an alternative routeto the pyruvate dehydrogenase reaction for the conversion of pyruvate toacetyl-CoA. The pyruvate dehydrogenase bypass comprises the enzymespyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) andacetyl-CoA synthetase (ACS). It has been shown in literature that thepyruvate dehydrogenase bypass is not essential in Crabtree-negativeyeasts.

In the present context the corresponding genes encoding the enzymeproteins are indicated in italics.

The term “PDC” refers to pyruvate decarboxylase enzyme (EC 4.1.1.1).This enzyme catalyses the thiamine pyrophosphate- and Mg²⁺-dependentdecarboxylation of pyruvate to acetaldehyde and carbon dioxide. Apreferred PDC is one of a Crabtree-positive organism. Preferably the PDCis a fungal PDC, especially of a Crabtree-positive fungus, such as S.cerevisiae e.g. PDC1 of S. cerevisiae (GenBank accession numberCAA54522, version number CAA54522.1). According to one embodiment of theinvention the PDC1 contains the amino acid sequence of SEQ ID NO:95,and/or is encoded for example by a polynucleotide containing thenucleotide sequence of SEQ ID NO:94, SEQ ID NO:96 or SEQ ID NO:97.

The term “ALD” refers to acetaldehyde dehydrogenase enzyme (EC 1.2.1.5and EC 1.2.1.4). This enzyme catalyses the reaction where acetaldehydeis oxidized to acetate, and NAD or NADP-cofactor is reduced to NADH orNADPH, respectively. NADP-specific ALDs are preferred. In the presentinvention the ALD is preferably a fungal ALD, more preferably of S.cerevisiae, and most preferably it is S. cerevisiae ALD6, which isencoded for example by a polynucleotide of SEQ ID NO:48 or 49, and/orcomprises the amino acid sequence of SEQ ID NO:47. Further, the ALD ispreferably a cytosolic ALD. ALD6 is cytosolic. Cytosolic ALD can bemodified from mitochondrial isoforms of ALD by removing themitochondrial targeting signal from the originally mitochondrial ALD bygenetic engineering. The cleavage site of the mitochondrial targetingsignal can be decided e.g. with programs designed for this purpose suchas MITOPROT. Examples of mitochondrial ALD, which can be modified to becytosolic are S. cerevisiae ALD4 and ALD5 encoding genes. Suitable ALDencoding genes can be found from databases e.g. KEGG Enzyme database andBrenda with EC numbers 1.2.1.4 and 1.2.1.5. Table 1 contains examples ofNAD(P)⁺ dependent acetaldehyde dehydrogenases.

TABLE 1 NAD(P)⁺ -dependent acetaldehyde dehydrogenase enzymes AccessionVersion number of the number of the amino acid amino acid Organismsequence sequence Database Saccharomyces cerevisiae AAB68304 AAB68304.1GenBank Saccharomyces cerevisiae DAA07732 DAA07732.1 GenBankSaccharomyces cerevisiae DAA11133 DAA11133.1 GenBank Aspergillus nigerA2QMA4 A2QMA4.1 TrEMBL Aspergillus niger A2QiG1 A2QiG1.1 TrEMBLAspergillus niger A5AAZ8 A5AAZ8.1 TrEMBL Aspergillus niger A2Q9V7A2Q9V7.1 TrEMBL Aspergillus fumigatus Q4WQP1 Q4WQP1.1 TrEMBL Pichiaangusta Q12648 Q12648 Swiss-Prot Pichia stipitis A3M013 A3M013.2 TrEMBLCandida dubliniensis B9W6J2 B9W6J2.1 TrEMBL Candida glabrata CAG59952CAG59952.1 GenBank Kluyveromyces lactis CAH00079 CAH00079.1 GenbankLachancea thermotolerans CAR23570 CAR23570.1 Genbank Burkholderiaxenovorans Q13WK4 Q13WK4.1 TrEMBL Vibrio harveyi Q56694 Q56694.1Swiss-Prot Mus musculus P47739 P47739.1 Swiss-Prot Mus musculus Q80VQ0Q80VQ0.1 Swiss-Prot Rattus norvegicus P11883 P11883.3 Swiss-Prot Rattusnorvegicus Q5XI42 Q5XI42.1 Swiss-Prot Canis lupus familiaris A3RF36A3RF36.1 Swiss-Prot Bos taurus P30907 P30907.2 Swiss-Prot Bos taurusQ1JPA0 Q1JPA0.1 Swiss-Prot Homo sapiens P30838 P30838.2 Swiss-Prot Homosapiens P43353 P43353.1 Swiss-Prot

The term “cytosolic” refers to a fluid component of the cytoplasmexcluding organelle and other suspended intracellular structures.

The term “ACS” refers to acetyl-CoA synthetase enzyme (EC 6.2.1.1). Theenzyme catalyses the reaction where acetyl-CoA is formed from acetateand CoA with hydrolysis of ATP. Preferably the ACS is a fungal ACS, morepreferably S. cerevisiae ACS, especially S. cerevisiae ACS2. Preferablythe ACS encoding gene to be expressed is a gene, which is not underglucose repression and/or which gene product is not subject topost-translational regulation e.g. acetylation, in the original species.In a particular embodiment the ACS contains the sequence of SEQ IDNO:50. Several yeasts such as Candida albicans have genes similar to S.cerevisiae ACS2, which most likely are not under post-translationalregulation. This abolishes the need to modify the existing gene.According to one embodiment ACS2 is encoded by a polynucleotide havingthe sequence of SEQ ID NO:51 or 92.

The term “DAT” refers to diacylglycerol acyltransferase enzyme (EC2.3.1.X). The enzyme catalyses a reaction where an acyl group istransferred to 1,2-diacylglycerol to the position sn-3. DAT includesboth acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) andphospholipid:diacylglycerol acyltransferase (PDAT). Preferably the DATis PDAT.

The term “PDAT” refers to phospholipid:diacylglycerol acyltransferaseenzyme (EC 2.3.1.158). The enzyme catalyses a reaction where an acylgroup from a phospholipid is transferred to 1,2-diacylglycerol to theposition sn-3 via an acyl-CoA-independent mechanism. Preferably the PDATis of fungal origin, and more preferably of an oleaginous fungus. In onepreferred embodiment the PDAT is a Rhizopus oryzae PDAT, and mostpreferably it is encoded by a polynucleotide that contains the sequenceof SEQ ID NO:53 or 93. Preferably the PDAT contains an amino acidsequence having at least 40% identity to SEQ ID NO:52, or anenzymatically active fragment or variant thereof.

In particular preferred embodiments of the invention the encoded enzymecomprises an amino acid sequence with a sequence identity of at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% to SEQ IDNO:95, 47, 50, or 52, or an enzymatically active fragment or variantthereof.

Percent identity of amino acid sequences can conveniently be computedusing BLASTP version 2.2.23 software with default parameters. Sequenceshaving an identities score and a positives score of a given percentage,using the BLASTP version 2.2.23 algorithm with default parameters, areconsidered to be that percent identical or homologous (Altschul et al.1997).

It is well known that deletion, addition or substitution of one or a fewamino acids does not necessarily change the catalytic properties of anenzyme protein. Therefore the invention also encompasses variants andfragments of the given amino acid sequences having the stipulated enzymeactivity. The term “variant” as used herein refers to a sequence havingminor changes in the amino acid sequence as compared to a givensequence. Such a variant may occur naturally e.g. as an allelic variantwithin the same strain, species or genus, or it may be generated bymutagenesis or other gene modification. It may comprise amino acidsubstitutions, deletions or insertions, but it still functions insubstantially the same manner as the given enzymes, in particular itretains its catalytic function as an enzyme.

A “fragment” of a given protein sequence means part of that sequence,i.e. a sequence that has been truncated at the N- and/or C-terminal end.It may for example be the mature part of a protein comprising a signalsequence, or it may be only an enzymatically active fragment of themature protein.

The term “lipid” refers to a group of organic compounds that arerelatively or completely insoluble in water but soluble in nonpolarorganic solvents. These properties are a result of long hydrocarbontails, which are hydrophobic in nature. The term thus encompasses fats,oils, waxes, fatty acids, fatty acid derivatives, like phospholipids,glycolipids, acylglycerids such as monoglycerides, diglycerides, andtriglycerides and terpenoids such as carotenoids and steroids.

The term “fatty acid” refers to a compound obtainable via condensationof malonyl coenzyme A units by a fatty acid synthase system. They may besaturated or unsaturated. “Functional fatty acid” refers to a fatty acidcompound having at least one functional group e.g. a hydroxyl (—OH) orcarboxyl (—COOH) group within the fatty acid and being responsible forthe characteristic chemical reactions of those molecules.

The term “fatty acid derivative” refers to a compound having at leastone esterified fatty acyl group. Fatty acid derivatives include e.g.phospholipids, glycolipids and acylglycerides.

The term “acylglyceride” is synonymous with “acylglycerol” and refers toa compound having a glycerol moiety with one or several hydroxyl groupsesterified to a fatty acid.

The terms “monoglyceride” and “monoacylglycerol” refer to a glyceridewhere one fatty acid residue has been esterified to a glycerol molecule.The fatty acid residue in the monoacylglycerol can be a short or longchain fatty acid with or without double bonds.

The terms “diglyceride” and “diacylglycerol” and “DAG” refer toglyceride where two fatty acid residues have been esterified to aglycerol molecule. Fatty acid residues in diacylglycerol can be short orlong chain fatty acids with or without double bonds.

The terms “triglyceride” and “triacylglycerol” and “TAG” refer to aglyceride where three fatty acid residues have been esterified to aglycerol molecule. Fatty acid residues in triacylglycerol can be shortor long chain fatty acids with or without double bonds. Triacylglycerolis the major acylglycerol group in oleaginous fungi.

The term “acyl-CoA” refers to a fatty acid residue, which has beenesterified to a CoA molecule. Fatty acid residues in acyl-CoA can beshort or long chain fatty acids with or without double bonds.

The term “phospholipids” refers to any lipid containing a diglyceridecombined with a phosphate group and a simple organic molecule such ascholine or ethanolamine.

The term “glycolipid” refers to a lipid attached with a carbohydrate.

The term “fat” refers to a group of organic compounds that arerelatively or completely insoluble in water but soluble in nonpolarorganic solvents and which are solids at normal room temperature.

The term “oil” refers to a group of organic compounds that arerelatively or completely insoluble in water but soluble in nonpolarorganic solvents and which are liquids at normal room temperature.

Generally fats and oils are triesters of glycerol and fatty acids.

The term “wax” refers to a compound that may contain long-chain alkanes,esters, polyesters and hydroxyl esters of long-chain primary alcoholsand fatty acids.

The term “terpenoid” refers to a compound formally derived fromhydrocarbon isoprene.

The term “steroid” refers to a terpenoid lipid compound having a steranecore and additional functional groups. Sterols are special forms ofsteroids, with a hydroxyl group at the atom C-3 and a skeleton derivedfrom cholestane.

The term “carotenoids” refers to a compound belonging to the category oftetraterpenoids. Structurally they are in the form of a polyene chain,which is sometimes terminated by rings.

In the present invention fungal cells are genetically modified toexpress particular enzymes. The cells can be genetically modified toproduce increased levels of lipid by transforming them with nucleicacids that have been modified to enhance the expression of nucleic acidsencoding at least one of PDC, ALD and ACS, together with a nucleic acidthat has been modified to enhance the expression of a nucleic acidencoding DAT so as to allow overexpression of the enzymes. A“genetically modified” organism or cell is an organism or cell thatcomprises an expression modified nucleic acid. It may be a recombinantorganism or cell, or a host organism or cell, or a mutant.

“Nucleic acid” is a macromolecule comprising a chain of monomericnucleotides i.e. a polynucleotide. It can be e.g. DNA such as cDNA orgenomic DNA or mRNA, and it can be e.g. recombinantly or syntheticallyproduced, double or single stranded, encompassing both sense andantisense strands.

“Recombinant” nucleic acid refers to an artificial combination of atleast two otherwise separated sequences, i.e. to a not naturallyoccurring combination of nucleic acids.

“Nucleic acid with modified expression” as used herein denotes nucleicacids that are foreign or exogenous to the host meaning that they arenot naturally found in said host. The term also includes nucleic acidsthat are endogenous i.e. naturally found in the host, but which areproduced in an unnatural amount e.g. as multiple copies, or nucleicacids that differ in sequence from the naturally occurring nucleic acidsbut encode the same type of protein. Further, the term includes nucleicacids comprising at least two nucleotide sequences that do not occur inthe same relationship to each other in nature, such as e.g. anendogenous protein encoding sequence that is operably linked to atranscriptional control element e.g. a promotor and/or terminator in away that does not occur in nature. Said promotor and/or terminator canbe of endogenous or exogenous origin. High copy number plasmidscomprising the protein encoding nucleotide sequence are also considerednucleic acids with modified expression. The above mentioned expressionmodified nucleic acids encompass recombinant nucleic acids, herein alsocalled heterologous nucleic acids. Alternatively the expression modifiednucleic acid can be a mutated nucleic acid. “Modified expression” inthis context is used only in the meaning of over-expression i.e.“enhanced expression”. The enhanced expression results in overproductionof the expressed protein in the modified organism compared to that in anunmodified organism.

In one embodiment of the invention the nucleic acid with modifiedexpression contains an exogenous gene encoding PDC, ALD, ACS or DATderived from another organism. The genes encoding PDC, ALD or ACS can beobtained from yeast such as Saccharomyces cerevisiae, Candida glabrata,Dekkera bruxellensis, Kluyvemmyces lactis, Kluyveromyces marxianus,Pichia pastoris, Pichia angusta, Pichia stipitis, Zygosaccharomycesrouxii, Issatchenkia terricola, Debaryomyces hansenii, Candida angustaand Pichia guilliermondii, and the DAT can be obtained from yeast orfilamentous fungi such as Saccharomyces cerevisiae, Candida glabrata,Zygosaccharornyces rouxii, Lachancea thermotolerans, Ashbya gossypii,Cryptococcus curvatus, Cryptococcus albidus, Lipomyces lipofer,Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides,Rhodotorula Rhodotorula graminis, Yarrowia lipolytica, Aspergillusnidulans, Aspergillus oryzae, Fusarium oxysporum, Humicola lanuginose,Mortierella alpina, Mortierella vinacea, Mucor circinelloides, Mucorplumbeus, Penicillium spinulosum, and Rhizopus oryzae. In anotherembodiment the enzyme genes to be expressed are endogenous genes, thepromotor of which is replaced with another promotor, preferably aconstitutive promotor, or the existing promotor is modified to become aconstitutive promotor.

Proteins or polynucleotides “derived from”, “originated from” or“obtained from” a particular organism encompass products isolated fromsaid organism, as well as modifications thereof. A protein derived froma particular organism may be a recombinantly produced product, which isidentical to, or a modification of the naturally occurring protein. Theprotein may also be modified e.g. by glycosylation, phosphorylation orother chemical modification. Products derived from the particularorganism also encompass mutants and natural variants of the products,where one or more nucleic acid and/or amino acid is deleted, insertedand/or substituted.

Expression of any combination of the genes of the pyruvate dehydrogenasebypass route may be linked to expression of the DAT encoding gene. Theexpression of the DAT encoding gene may for example be combined to theexpression of an ALD encoding gene, or an ACS encoding gene, or a PDCencoding gene. In another embodiment both ASC and ALD, or both PDC andACS, or PDC and ALD, are overexpressed, and in still another all PDC,ALD and ACS are overexpressed together with the DAT encoding gene. Inone specific embodiment of the invention the expression of S. cerevisiaeALD encoding gene ALD6 is linked to the expression of a PDAT encodinggene.

Alternatively expression of PDC may be combined with expression of ACSand/or ALD thus providing the combinations PDC+ALD, PDC+ACS andPDC+ALD+ACS. These combinations may be expressed with or without furtherexpressing a gene encoding DAT, such as PDAT.

“Fungal” “fungus” and “fungi” as used herein refers to yeast andfilamentous fungi i.e. moulds. A genetically modified fungal cell isalso referred to as host cell.

The yeast host cell may be selected for example from the generaCryptococcus, Candida, Galactomyces, Hansenula, Lipomyces,Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia. Preferably theyeast host cell is selected from the group consisting of Candida sp.,Cryptococcus curvatus, Cryptococcus albidus, Galactomyces geotrichum,Hansenula ciferri, Lipomyces hpofer, Lipomyces ssp., Lipomyces starkeyi,Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis,Trichosporon pullulans and Yarrowia lipolytica. In one embodiment theyeast is selected from the phylum Basidiomycota, which includesCryptococcus, Rhodotorula and Rhodosporidium. Most preferably it isCryptococcus curvatus.

The filamentous fungus host cell may be selected from the generaAspergillus, Cunninghamella, Fusarium, Glomus, Humicola, Mortierella,Mucor, Penicillium, Pythium and Rhizopus. Preferably the filamentousfungus is selected from the group consisting of Aspergillus nidulans,Aspergillus oryzae, Aspergillus terreus, Aspergillus niger,Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius, Humicolalanuginose, Mortierella isabellina, Mortierella pusilla, Mortierellavinacea, Mucor circinelloides, Mucor plumbeus, Mucor ramanniana,Penicillium lilacinum, Penicillium spinulosum, Pythium ultimum andRhizopus oryzae. According to one embodiment the filamentous fungusbelongs to subphylum Mucoromycotina, which includes Mucor andMortierella. Most preferably it is Mucor circinelloides.

According to one preferred embodiment the fungal host cell is anoleaginous fungus. The term “Oleaginous fungi” refers to yeasts orfilamentous fungi, which accumulate at least 10%, 12.5%, 15%, 17.5%,preferably at least 20% or even at least 25% (w/w) of their biomass aslipid. They may even accumulate at least 30%, 40%, 50%, 60%, 70%, 80%(w/w) or more of their biomass as lipids. The biomass is usuallymeasured as cell dry weight (CDW). Oleaginous fungi are found e.g. ingenera Cryptococcus, Candida, Galactomyces, Hanseluna, Lipomyces,Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia, Aspergillus,Cunninghamella, Fusarium, Glomus, Humicola, Mortierella, Mucor,Penicillium, Pythium and Rhizopus, and especially in species Candidasp., Cryptococcus curvatus, Cryptococcus albidus, Galactomycesgeotrichum, Hansenula ciferri, Lipomyces lipofer, Lipomyces ssp.,Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides,Rhodotorula glutinis, Trichosporon pullulans, Yarrowia lipolytica,Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus,Aspergillus niger, Cuninghamella japonica, Fusarium oxysporum, Glomuscaledonius, Humicola lanuginose, Mortierella isabellina, Mortierellapusilla, Mortierella vinacea, Mucor circinelloides, Mucor plumbeus,Mucor ramanniana, Penicillium lilacinum, Penicillium spinulosum, Pythiumultimum and Rhizopus oryzae. In one embodiment it is a Crabtree-negativeoleaginous yeast, and in another embodiment it is a Crabtree-positivefilamentous fungus. In still another embodiment the filamentous fungusis Crabtree-negative, or the yeast is Crabtree-positive. Saccharomycesyeasts including S. cerevisiae are not oleaginous fungi. A“Crabtree-positive” organism is one that is capable of producing ethanolin the presence of oxygen, whereas a “Crabtree-negative” organism isnot.

According to one preferred embodiment the host cell is a Cryptococcus,and especially Cryptococcus curvatus. Genetically modified Cryptococcuscurvatus strains have not been described in literature (Meesters et al.1997). Routinely, in yeast expression systems S. cerevisiae promotersand terminators, original genes without codon optimisation orcodon-optimised for S. cerevisiae are used. In this invention we showedthat it is possible to express genes successfully in C. curvatus whenusing endogenous promoters and terminators i.e. which originate from thespecies wherein the expression cassette will be transformed, and anexpressed gene that is codon-optimised according to codon usage of thespecies wherein the expression cassette will be transformed, or itsclose relative, which has a genome that is known at a level wherecodonoptimisation is possible. Such a close relative is e.g. Ustilagomaydis. In a preferred embodiment the promotors are constitutivepromotors, especially from the glycolysis pathway.

Promoters and terminators of oleaginous fungi might contain sites fordifferent regulatory elements and transcription factors than thepromoters and terminators of S. cerevisiae due to the different natureof the strains: S. cerevisiae can grow and produce ethanol underanaerobic conditions whereas C. curvatus does not produce ethanol atall, and lipids it produces under aerobic conditions. Also the codonusage differs in S. cerevisiae and in C. curvatus: E.g. S. cerevisiaecodons and genome are adenine and thymine rich, whereas the ratio ofguanine and cytosine is much higher in C. curvatus (Meesters et al.1997).

It is also important to use endogenous promoters and terminators withresistance markers to detect transformants after transformations. Due tothe fact that the genome of C. curvatus is not known differentprocedures described in this invention can be carried out to cloneendogenous promoters and terminators. Preferably constitutive promotersare used.

The fungal cell can be genetically modified by transforming it with aheterologous nucleic acid that encodes a heterologous protein.“Heterologous” in this context means not naturally occurring. In oneembodiment, the cell is transformed with a heterologous nucleic acidthat encodes at least one of PDC, ALD and ACS, and a heterologousnucleic acid comprising a nucleic acid that encodes DAT, operably linkedto allow expression of the genes encoding said enzymes. DNA isolation,enzymatic treatment and genetical modifications may be carried out usingstandard molecular biology methods as described e.g. Sambrook andRussell (2001). The promoter and terminator regions of the genes ofinterest can be cloned e.g. from a yeast or filamentous fungus strain ofinterest by polymerase chain reaction (PCR) using gene specificoligonucleotides designed based on known published gene sequences ofstrains of the same species or other species or gene sequences of thestrain of interest. Oligonucleotides designed based on the sequence ofthe strain of interest are preferred.

New gene fragments from yeast and filamentous fungus species or strainswith unknown genomic sequences can be cloned by PCR by usingdegenerative primers. The term “degenerative primer” refers to mixturesof similar kinds of synthesized primers differing from each other by onenucleotide. Degenerative primers for cloning of specific gene fragmentsfrom desired species or strains are designed based on knowncharacterised or putative gene sequences.

A person skilled in art can use known characterised gene sequences astemplates in a Blast search to find out other characterised or putativegene sequences. Another possibility is to search specific gene sequencesby enzymes names from databases containing genomic sequences of speciesfrom different genome sequencing projects. These kinds of databases arefound for example, but not excluding, in Broad Institute's Fungal GenomeInitiative sequence projects. In the searches of specific gene sequencesspecies that are closely related to the yeast and filamentous fungusspecies of interest are preferred. After a set of specific genesequences has been found nucleotide sequence alignments are carried outwith appropriate programs e.g. Clustal W, resulting in a consensussequence, which is used in designing degenerative primers. Designeddegenerative primers are used in a PCR reaction with DNA of the yeast orfilamentous fungus strain of interest as a template. Resulting PCRfragments are gel isolated and sequenced directly or after being clonedinto plasmids. Detected sequences are used in Blast searches to confirmthat right gene fragments have been cloned.

An unknown promoter and/or terminator region of a gene of interest canbe cloned by a chromosome walking method. The term “chromosome walking”refers to sequential isolation of clones carrying overlapping sequencesof a known gene region and an unknown sequence of an adjacent generegion produced by ligation-mediated PCR amplification method (Muellerand Wold 1989). Gene specific oligonucleotides corresponding to a knownsequence of a gene of interest are designed and used in PCR reactionswith linker specific oligonucleotides. The known sequence of the gene ofinterest may originate e.g. from a sequence of a gene fragment generatedin a PCR reaction with degenerative oligos or from a gene sequencepublished in sequence databases. The resulting PCR fragments are gelisolated and sequenced directly or after being cloned into plasmids.Detected sequences are used in Blast searches to confirm that right genefragments have been cloned. If needed, the chromosome walking experimentwill be repeated so many times that desired length of promoter orterminator region has been cloned. The existence of the sequence of thepromoter or terminator region of the desired gene is confirmed by usualbioinformatics methods e.g. with multiple sequence alignment.

A strain specific gene fragment containing the promoter and/orterminator region of the gene of interest can also be cloned byconventional library screening methods described e.g. in Sambrook andRussell (2001). The sequences of the oligonucleotides used in PCRreactions to clone desired promoter or terminator regions can alsocontain sequences of restriction sites of specific restriction enzymesin addition to the gene specific sequence. The PCR fragment containingthe desired promoter or terminator will be cloned into plasmid e.g.pBluescript and sequenced.

Promoters used in expression cassettes can be promoters ofconstitutively expressed genes e.g. of 3-phosphoglycerate dehydrogenase(PGK), triose phosphate isomerase (TPI) or enolase (ENO). Alternatively,the promoter used in the expression cassette can be a promoter of agene, which is expressed under specific cultivation conditions.

“Expression cassette” as used herein refers to a nucleic acid constructthat comprises a transcription initiation or transcription controlsequence, e.g. a promotor, operably linked to a coding region for theprotein to be transcribed, and preferably a transcription terminationregion. In addition it conveniently comprises one or more markerregions, i.e. regions encoding a selection marker.

Genes to be expressed can be cloned directly from the desired species orstrains by conventional molecular biology methods e.g. by using PCR.More preferably the genes to be expressed are synthesised using a codonoptimised nucleotide sequence based on the known codon usage of the hoststrain. If the codon usage of the host strain or host species is notknown, the gene to be expressed can be codon optimised based on theknown codon usage of a closely related species of the host strain.Alternatively, the gene to be expressed can be synthesised according toa known amino acid sequence by translating the amino acid sequence intoa DNA sequence, preferably into a codon-optimised DNA sequence. The term“codon optimization” refers to an optimization of a synthetic nucleotidesequence encoding expressed genes to enhance gene product production ina host strain. Codon optimization can occur by replacing existing codonsof the original gene by the codons used more often in the host strain.In codon optimisation also internal TATA-boxes, chi-sites, ribosomalentry sites, AT-rich or GC-rich sequence stretches, repeated sequences,RNA secondary structures and cryptic splice donor and acceptor sites canbe avoided. Additionally in codon optimisation sequences of restrictionsites of specific restriction enzymes can be avoided. Preferably, in asynthesised gene sequence the CTG codon will be avoided due to itsdifferent coding in different fungi (leucine or serine).

A nucleic acid that is “degenerate as a result of the genetic code” to agiven sequence, means that it contains one or more different codons, butencodes for the same amino acids. A “polynucleotide” as used herein maybe a single or double stranded polynucleic acid. The term encompassesgenome DNA, cDNA and RNA.

A “synthetic gene” or “synthetic nucleotide sequence” is an artificiallydesigned gene or sequence, which has been synthesised into a physicalDNA sequence.

The gene to be expressed is cloned between a promoter and terminator. Anexpression cassette of the gene to be expressed with promoter andterminator, and a marker gene can be transformed. The marker gene can beunder its own promoter and terminator, but preferably it is under afunctional promoter and terminator from another species, or morepreferably under a functional promoter and terminator of the hoststrain. Markers to be used can be antibiotic markers like genes forhygromycin, geneticin and cerulenin resistances or other dominant markerlike the melibiase gene. Additionally genes of the amino acid synthesiscan be used as markers with auxotrophic fungi. The gene to be expressedcan be transformed into the yeast or filamentous fungus as a plasmid toproduce epitopic transformants, or as a DNA fragment containing theexpression cassette to produce transformants with the expressioncassette integrated into the genome of the host strain.

Expression cassettes containing the marker gene and gene to be expressedcan be transformed in the same DNA fragment, or the expression cassettesof marker gene and gene to be expressed can be in separate DNAfragments. Transformation methods contain chemical, protoplast,electroporation methods. Transformants can be selected based on theirability to grow on a medium (solid or liquid) containing antibiotics, ora medium lacking some essential component e.g. an amino acid, ortransformants can be selected based on different phenotype such ascolour reaction of the transformants in specific conditions.

The DNA level of the transformants can be characterised by PCR or bySouthern analysis using conventional molecular biology methods.Additionally enzyme activities of the expressed gene can be assayed asindicated in the example 27 or as described e.g. in Dahlqvist et al.2000 and Postma et al. 1989,

The gene to be expressed can be an endogenous gene, whereby the promoterregion can be replaced with a promoter of a constitutively expressedgene, or with a promoter of a gene, which is expressed under specificcultivation conditions. Additionally, expression of an endogenous genecan be enhanced by classical mutagenesis.

The genetically modified fungi of the present invention are capable ofproducing increased levels of lipids, and especially oftriacylglycerols. The increase may be at least a 1.5, 3, 5 or 10 foldincrease in lipid or triacylglycerol concentration in transformantscompared to the unmodified host strain during cultivation.Alternatively, it may be at least a 1.5, 3, 5 or 10 fold increase inlipid or triacylglycerol yield per used carbon source (e.g. glucose) intransformants compared to the unmodified host strain. It may also referto a 1.5, 3, 5 or 10 fold increase in lipid or triacylglycerolproduction rate (mg/l/h) compared to the unmodified host strain. Thisincrease in lipid or triacylglycerol production can be detected eitherintracellularly or in the amount of lipids and triacylglycerols inculture medium.

The genetically modified fungi are cultivated in a medium containingappropriate carbon and nitrogen sources together with other optionalingredients like yeast extract, peptone, minerals and vitamins, such asKH₂PO₄, Na₂HPO₄, MgSO₄, CaCl₂, FeCl₃, ZnSO₄, citric acid, MnSO₄, CoCl₂,CuSO₄, Na₂MoO₄, FeSO₄, H₃BO₄, D-biotin, CaPantothenate, nicotinic acid,myoinositol, thiamine, pyridoxine, p-aminobenzoic acid. The inventionworks at a wide range of C/N ratios about from 20 to 160 undermicroaerobic (50 ml medium in 250 ml flask with 100 rpm shaking) andaerobic (50 ml medium in 250 ml flask with 250 rpm shaking or 1-2 vvm inbioreactors) conditions from the beginning of the cultivation to the endof cultivation as far as up to at least 8 days. The host cells used arepreferably such that are able not only to use hexoses, such as glucose,but also pentoses such as xylose, and arabinose, or even glycerol ascarbon source. Preferably the carbon source is a hexose and/or pentosesugars containing material such as cellulose or hemicellulose. Thegenetically modified host cells are preferably grown on agricultural orindustrial waste materials e.g. cellulose or hemicellulose containingmaterials, which makes the lipid production economically andenvironmentally beneficial.

After cultivation, the yeast or filamentous fungal cells are normallyseparated from the culture medium. Lipids are recovered from the cellstypically by using non-polar organic solvents, such as hexane. Prior toextraction, cells can be dried and/or disrupted. Alternatively, thelipids can be recovered directly from the culture medium, which ofcourse is an advantage. This is especially convenient when the host cellis Cryptococcus, such as C. curvatus. Preferably lipid extraction iscarried out to obtain lipids which mainly contain triacylglycerol (TAG).The lipid fraction can also contain mono- and diglycerides, free fattyacids, phospholipids, glycolipids and other lipids.

The lipids, and especially the TAG and the fatty acids, obtained can beused for preparing biofuels and lubricants. Said lipids may be directlyused as biofuel or lubricant, but usually they are further processed toe.g. biodiesel or renewable diesel and/or lubricant formulations.“Biofuel” as used herein refers to fuel that has been at least partiallybiologically produced.

“Biodiesel” consists essentially of fatty acid methyl esters and istypically produced by transesterification in which the acylglyceridesare converted to fatty acid methyl esters. According to EU directive2003/30/EU biodiesel refers to a methyl-ester produced from vegetableoil or animal oil, of diesel quality to be used as biofuel. Morebroadly, biodiesel refers to long-chain alkyl esters, such as methyl,ethyl or propyl esters, from vegetable oil or animal fat of dieselquality. In the present content biodiesel can also be produced fromfungal lipids.

“Renewable diesel” refers to fuel which is produced by hydrogentreatment (hydrogenation or hydroprocessing) of lipids of animal,vegetable or fungal origin, or their mixtures. Renewable diesel can beproduced also from waxes derived from biomass by gasification andFischer-Tropsch synthesis. Optionally, in addition to hydrogentreatment, isomerization or other processing steps can be performed. Inhydrogen treatment, acylglycerides are converted to correspondingalkanes (paraffins). The alkanes (paraffins) can be further modified byisomerization or by other process alternatives. These processes can alsoproduce hydrocarbons which are suitable for jet fuel or gasolineapplications.

“Lubricant” refers to a substance, such as grease, lipid or oil thatreduces friction when applied as a surface coating to moving parts. Twoother main functions of a lubricant are heat removal and dissolvingimpurities. Applications of lubricants include, but are not limited touses in internal combustion engines as engine oils, additives in fuels,in oil-driven devices such as pumps and hydraulic equipment, or indifferent types of bearings. Typically lubricants contain 75-100% baseoil and the rest is additives. In the present invention at least part ofthe base oil is of fungal origin. Viscosity index is used tocharacterise base oil. Typically high viscosity index is preferred.

Lubricants or at least the base oil for lubricants may be prepared inthe same way as described above for the biofuels. Usually conventionaladditives such as viscosity modifyers, antioxidants, pour pointmodifyers etc. are added to the base oil to obtain the lubricant.

The lipids, and especially the TAG and the fatty acids obtained can alsobe used for precursors for functional fatty acids. Said lipids arefurther processed to release fatty acyls which can be used in theproduction of functional fatty acids, like dicarboxylic acids andepoxides, which can be further converted into products like polyesters,polyurethane, coatings and resins.

The following examples are provided to illustrate the invention, but arenot intended to limit the scope thereof. The enzyme names used are basedon sequence homology.

Example 1A: Cloning of Cryptococcus curvatus TEF (CcTEF1) Promoter andTerminator Region

A genomic ˜800 bp fragment of the C. curvatus TEF gene was amplified byPCR with degenerative primers identified as SEQ ID NO:1 (Yeast TEF1),and SEQ ID NO:2 (Yeast TEF4), using C. curvatus (C-01440, VTT CultureCollection) genomic DNA as the template. The degenerative primers weredesigned based on a consensus sequence of the putative TEF1 genes ofUstilago maydis, Candida guilliermondii and Candida tropicalis. Thedetected genomic fragment was sequenced.

Genomic fragments containing the CcTEF1 promoter region were obtainedwith ligation-mediated PCR amplification (Mueller, P. R. and Wold, B.1989). A mixture of a linker identified as SEQ ID NO:3 (PCR linker I),and a linker identified as SEQ ID NO:4 (PCR linker II) was ligated toPvuII digested C. curvatus genomic DNA with T4 DNA ligase (New EnglandBioLabs). Samples of the ligation mixtures were used as templates for 50μl PCR reactions containing 0.1 μM of a primer identified as SEQ ID NO:3 (PCR linker I), and 1 μM of a primer identified as SEQ ID NO:5(CC_TEF2). The reaction mixture was heated at 94° C. for 3 minutes after2 U of Dynazyme EXT was added. The reactions were cycled 30 times asfollows: 1 minute at 94° C., 2 minutes at 60° C. and 2 minutes at 72°C., with final extension of 10 minutes at 72° C. A diluted sample ofthis first PCR-amplification was used as the template in a nested PCRreaction (50 μl) containing 0.05 μM of a primer identified as SEQ IDNO:3 (PCR Linker I), and 0.5 μM of a primer identified as SEQ ID NO:6(CC_TEF1). The reaction mixture was heated at 94° C. for 3 minutes after2 U of Dynazyme EXT was added. The reactions were then cycled 30 timesas follows: 1 minute at 94° C., 2 minutes at 60° C. and 2 minutes at 72°C., with final extension of 10 minutes at 72° C.

A ˜800 bp fragment was isolated and sequenced. Nested primers identifiedas SEQ ID NO:7 (CC_TEF6), and SEQ ID NO:8 (CC_TEF5) were designed andused in a ligation-mediated PCR amplification together witholigonucleotides identified as SEQ ID NO:3 (PCR linker I), and a linkeridentified as SEQ ID NO:4 (PCR linker II) similarly as above except thatNruI-digested C. curvatus DNA was used. A ˜2000 bp PCR fragment wasisolated and sequenced.

The C. curvatus TEF1 promoter was PCR amplified using primers identifiedas SEQ ID NO:9 (CC_TEF10), and SEQ ID NO:10 (CC_TEF11) and the C.curvatus genomic DNA as the template. A PCR fragment was digested withSacII and XbaI. A 1276 bp fragment was gel isolated and ligated to aSacII and XbaI-digested pBluescript KS-plasmid (Stratagene). Theresulting plasmid was designated pKK58. Plasmid pKK58 contains C.curvatus TEF1 promoter.

A genomic fragment containing the CcTEF1 terminator region was obtainedwith a ligation-mediated PCR amplification with C. curvatus TEF1 genespecific oligonucleotides identified as SEQ ID NO:11 (CC_TEF3), and SEQID NO:12 (CC_TEF4) together with oligonucleotides identified as SEQ IDNO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II) similarly as aboveexcept that NruI-digested C. curvatus DNA was used. A ˜1600 bp PCRfragment was isolated and sequenced.

The C. curvatus TEF1 terminator was PCR amplified using primersidentified as SEQ ID NO:13 (CC_TEF7) and SEQ ID NO:14 and the C.curvatus genomic DNA as the template. A PCR fragment was digested withXmaI and EcoRI. A 358 bp fragment was gel isolated and ligated to XmaIand EcoRI-digested pBluescript KS-plasmid. The resulting plasmid wasdesignated pKK55. Plasmid pKK55 contains the C. curvatus TEF1terminator.

Example 1B: Cloning of Cryptococcus curvatus TPI (CcTPI1) Promoter andTerminator Region

A genomic fragment of the C. curvatus TPI1 gene was amplified by PCRfrom genomic C. curvatus (C-01440, VTT Culture Collection) DNA withdegenerative primers identified as SEQ ID NO:15 (Yeast TPI5), and SEQ IDNO:16 (Yeast TPI8). The degenerative primers were designed based on aconsensus sequence of the TPI1 genes of Ustilago maydis and Cryptococcusneoformans. A ˜800 bp genomic fragment was isolated and sequenced.

A genomic fragment containing the CcTPI1 promoter region was obtainedwith a ligation-mediated PCR amplification with TPI1 gene specificoligonucleotides identified as SEQ ID NO:17 (CC_TPI2), and SEQ ID NO:18(CC_TPI1), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that EcoRV-digested C. curvatus DNA was used. A ˜1300 bp PCRfragment was isolated and sequenced.

The C. curvatus TPI1 promoter was PCR amplified by using primersidentified as SEQ ID NO:19 (CC_TPI7) and SEQ ID NO:20 (CC_TPI_9) and theC. curvatus DNA as the template. A PCR fragment was digested with BamHIand SbfI. A 851 bp fragment was gel isolated and ligated to a BamHI andPstI-digested pBluescript KS-plasmid. The resulting plasmid wasdesignated pKK63. Plasmid pKK63 contains the C. curvatus TPI1 promoter.

A genomic fragment containing the CcTPI1 terminator region was obtainedwith a ligation-mediated PCR amplification with TPI1 gene specificoligonucleotides identified as SEQ ID NO:21 (CC_TPI 4) and SEQ ID NO:22(CC_TPI3), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that EcoRV-digested C. curvatus DNA was used. A ˜1200 bp PCRfragment was isolated and sequenced.

The C. curvatus TPI1 terminator was PCR amplified by using primersidentified as SEQ ID NO:23 (CC_TPI5) and SEQ ID NO:24 (CC_TPI6) and theC. curvatus genomic DNA as the template. A PCR fragment was digestedwith XbaI and BamHI. A 361 bp fragment was gel isolated and ligated to aXbaI and BamHI-digested pBluescript KS-plasmid. The resulting plasmidwas designated pKK61. Plasmid pKK61 contains C. curvatus TPI1terminator.

Example 1C: Cloning of Cryptococcus curvatus ENO (CcENO1) Promoter andTerminator Region

A genomic fragment of the C. curvatus ENO1 gene was amplified by PCRfrom genomic C. curvatus (C-01440, VTT Culture Collection) DNA withdegenerative primers identified as SEQ ID NO:25 (YeastENO5) and SEQ IDNO:26 (YeastENO10). The degenerative primers were designed based on aconsensus sequence of ENO1 genes of Ustilago maydis and Cryptococcusneoformans. A ˜1000 bp genomic fragment was isolated and sequenced.

A genomic fragment containing the CcENO1 promoter region was obtainedwith a ligation-mediated PCR amplification with ENO1 gene specificoligonucleotides identified as SEQ ID NO:27 (CC_ENO2) and SEQ ID NO:28(CC_ENO1), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that PvuII-digested C. curvatus DNA was used.

A ˜600 bp fragment was isolated and sequenced. Nested primers identifiedas SEQ ID NO:29 (CC_ENO5) and SEQ ID NO:30 (CC_ENO6) were designed andused in a ligation-mediated PCR amplification together witholigonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ IDNO:4 (PCR Linker II) similarly as above except that SspI-digested C.curvatus DNA was used. A 2000 bp PCR fragment was isolated andsequenced.

The C. curvatus ENO1 promoter was PCR amplified by using primersidentified as SEQ ID NO:31 (CC_ENO9) and SEQ ID NO:32 (CC_ENO10) and theC. curvatus genomic DNA as the template. A PCR fragment was digestedwith EcoRI. A 1214 bp fragment was gel isolated and ligated to aEcoRI-digested pBluescript KS-plasmid. The resulting plasmid wasdesignated pKK74. Plasmid pKK74 contains the C. curvatus ENO1 promoter.

A genomic fragment containing the CcENO1 terminator region was obtainedwith a ligation-mediated PCR amplification with ENO1 gene specificoligonucleotides identified as SEQ ID NO:33 (CC_ENO4) and SEQ ID NO:34(CC_ENO3), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that NruI-digested C. curvatus DNA was used. A ˜1100 bp PCRfragment was isolated and sequenced.

The C. curvatus ENO1 terminator was PCR amplified by using primersidentified as SEQ ID NO:35 (CC_ENO7) and SEQ ID NO:36 (CC_ENO8), and theC. curvatus genomic DNA as the template. A PCR fragment was digestedwith HindIII. A 375 bp fragment was gel isolated and ligated to aHindIII-digested pBluescript KS-plasmid. The resulting plasmid wasdesignated pKK60. Plasmid pKK60 contains the C. curvatus ENO1terminator.

Example 1D: Cloning of C. curvatus GPD (CcGPD1) Terminator Region

A genomic fragment containing the CcGPD1 terminator region was obtainedwith a ligation-mediated PCR amplification with GPD1 gene specificoligonucleotides identified as SEQ ID NO:37 (CC_GPD3) and SEQ ID NO:38(CC_GPD4), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that SspI-digested C. curvatus DNA was used. A ˜1800 bp fragmentwas isolated and partially sequenced. GPD1 gene specificoligonucleotides were designed according to the C. curvatus GPD1 gene(GenBank Accession number AF126158, version number AF126158.1) sequence.

The C. curvatus GPD1 terminator was PCR amplified by using primersidentified as SEQ ID NO:39 (CC_GPD6) and SEQ ID NO:40 (CC_GPD7), and theC. curvatus genomic DNA as the template. A PCR fragment was digestedwith XbaI and BamHI. A 336 bp fragment was gel isolated and ligated to aXbaI and BamHI-digested pBluescript KS-plasmid. The resulting plasmidwas designated pKK54. Plasmid pKK54 contains C. curvatus GPD1terminator.

Example 2A: Cloning of the E. coli Hygromycin Resistance Gene;Construction of a Plasmid (pKK76) Having the E. Con HygromycinResistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1Terminator

The E. coli hygromycin (hph) gene, that confers resistance to hygromycinB, was PCR amplified using primers identified as SEQ ID NO:41 (Hph 5)and SEQ ID NO:42 (Hph 3), and the plasmid pRLMEX30 (Mach et al. 1994)DNA as the template. A PCR fragment was digested with SpeI. A 1048 bpfragment was gel isolated and ligated to SpeI-digested pBluescriptKS-plasmid and sequenced. The resulting plasmid was designated pKK52.

Plasmid pKK58 was digested with SacII and SbfI. A 1272 bp fragment wasgel isolated. Plasmid pKK52 was digested with SbfI. A 1034 bp fragmentwas gel isolated. Plasmid pKK58 contains the C. curvatus TEF1 promoterand plasmid pKK61 contains the C. curvatus TPI1 terminator. The 1272 bpfragment originating from plasmid pKK58 and the 1034 bp fragmentoriginating from the pKK52 plasmid were ligated to a 3285 bp fragmentobtained by digesting a plasmid designated as pKK61 with SacII and SbfI.The resulting plasmid was designated pKK76. Plasmid pKK76 contains theE. coli hygromycin gene under the control of the C. curvatus TEF1promoter and the C. curvatus TPI1 terminator.

Example 2B: Cloning of the E. coli G418 Resistance Gene; Construction ofa Plasmid (pKK67) Having the E. coli Geneticin Resistance Gene Under theControl of the CcTEF1 Promoter and the CcTPI1 Terminator

The E. coli G418 resistance gene was PCR amplified using primersidentified as SEQ ID NO:43 (Kan 5) and SEQ ID NO:44 (Kan 3), and theplasmid pPIC9K (Invitrogen) DNA as the template. A PCR fragment wasdigested with SpeI. A 838 bp fragment was gel isolated and ligated toSpeI-digested pBluescript KS-plasmid and sequenced. The resultingplasmid was designated pKK51.

Plasmid pKK58 was digested with SacII and SbfI. A 1272 bp fragment wasgel isolated. Plasmid pKK51 was digested with SbfI. A 824 bp fragmentwas gel isolated. The 1272 bp fragment originating from pKK58 plasmidand the 824 bp fragment originating from pKK51 plasmid were ligated to a3285 bp fragment obtained by digesting a plasmid designated pKK61 withSacII and SbfI. Plasmid pKK58 contains the C. curvatus TEF1 promoter andplasmid pKK61 contains the C. curvatus TPI1 terminator. The resultingplasmid was designated pKK67. Plasmid pKK67 contains the E. coli G418resistance gene under the control of the C. curvatus TEF1 promoter andthe C. curvatus TPI1 terminator.

Example 2C: Cloning of S. cerevisiae Cerulenin Resistance Gene;Construction of a Plasmid (pKK91) Having the S. cerevisiae CeruleninResistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1Terminator

The S. cerevisiae cerulenin resistance gene was PCR amplified usingprimers identified as SEQ ID NO:45 (CERR 5) and SEQ ID NO:46 (CERR 3),and the plasmid pSH47Y DNA as the template. Plasmid pSH47Y contains thecerulenin resistance gene from the plasmid pCR1 (Nakazawa et al. 1993).A PCR fragment was digested with SbfI. A 1685 bp fragment was gelisolated and ligated to PstI-digested pBluescript KS-plasmid andsequenced. The resulting plasmid was designated pKK80.

Plasmid pKK80 was digested with PstI. A 1685 bp fragment was gelisolated and ligated to a 4557 bp fragment obtained by digesting aplasmid designated as pKK67 with SbfI. Plasmid pKK67 contains the E.coli G418 resistance gene linked to a C. curvatus TEF1 promoter and C.curvatus TEF1 terminator. The resulting plasmid was designated pKK91.Plasmid pKK91 contains the S. cerevisiae cerulenin resistance gene underthe control of the C. curvatus TEF1 promoter and the C. curvatus TPI1terminator.

Example 3A. Construction of a Plasmid (pKK81, FIG. 3) Containing theHygromycin Resistance Gene Under the Control of the CcTEF1 Promoter andthe CcTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under theControl of the CcENO1 Promoter and the CcTEF1 Terminator

The plasmid UmALD (Geneart AG, Germany) contains a S. cerevisiae ALD6(SEQ ID NO:47) encoding gene which has been codon optimized according toUstilago maydis yeast codon usage (SEQ ID NO:48) with flanking SbfIrestriction sites. The plasmid RoALD (Geneart AG, Germany) contains a S.cerevisiae ALD6 (SEQ ID NO:47) encoding gene which has been codonoptimized according to Rhizopus oryzae filamentous fungus codon usage(SEQ ID NO:49) with flanking SbfI restriction sites and an E. colikanamycin resistance gene. Plasmid UmALD was digested with SfiI. A 1554bp fragment was gel isolated and ligated to a 2258 bp fragment obtainedby digesting a plasmid RoALD with SfiI. The resulting plasmid wasdesignated as pKK50. The plasmid pKK50 contains a S. cerevisiae ALD6(SEQ ID NO:47) encoding gene which has been codon optimized according toUstilago maydis yeast codon usage (SEQ ID NO:48) with flanking SbfIrestriction sites and an E. coli kanamycin resistance gene.

Plasmid pKK74 was digested with EcoRI and SbfI. A 1204 bp fragment wasgel isolated. Plasmid pKK55 was digested with EcoRI and SbfI. A 347 bpfragment was gel isolated. The 1204 bp fragment originating from plasmidpKK74 and the 347 bp fragment originating from plasmid pKK55 wereligated to a 2961 bp fragment obtained by digesting a plasmid designatedpKK74 with EcoRI. Plasmid pKK74 contains C. curvatus ENO1 promoter andplasmid pKK55 contains C. curvatus TEF1 terminator. The resultingplasmid was designated as pKK77pre.

Plasmid pKK50 was digested with SbfI. A 1514 bp fragment was gelisolated and ligated to a 4517 bp fragment obtained by digesting plasmidpKK77pre with SbfI. The resulting plasmid was designated as pKK77. Theplasmid pKK77 contains the S. cerevisiae ALD6 encoding gene under thecontrol of the CcENO1 promoter and the CcTEF1 terminator.

Plasmid pKK76 was digested with BamHI and XmnI. A 2652 bp fragment wasgel isolated and ligated to a 6031 bp fragment obtained by digestingplasmid pKK77 with BamHI. The plasmid pKK76 contains the E. colihygromycin resistance gene under the control of the CcTEF1 promoter andthe CcTPI1 terminator. The resulting plasmid was designated as pKK81(FIG. 3).

Example 3B. Generation of a Genetically Modified C. curvatus (Y23/81)with an Integrated ALD6 Encoding Gene and Hygromycin Resistance Gene byTransforming Wild-Type C. curvatus with Digested Plasmid pKK81 (FIG. 3,Ex. 3A)

Plasmid pKK81 was restricted with NotI and PspOMI, and the resultinglinear DNA was used to transform a wild-type C. curvatus strain ATCC20509 designated as Y23 by electroporation using a standardelectroporation method.

The transformed cells were screened for hygromycin resistance. Severalhygromycin-resistant colonies were analysed at DNA level by PCR. Thetransformants originating from the transformation of C. curvatus withNotI and PspOMI cut pKK81 and containing a S. cerevisiae ALD6 encodinggene under the control of the CcENO1 promoter and the CcTEF1 terminatorwere designated as Y23/81-8, Y23/81-51, Y23/81-59, Y23/81-66 andY23/81-69.

Example 4A. Construction of a Plasmid (pKK82, FIG. 4) Containing theG418 Resistance Gene Under the Control of the CcTEF1 Promoter and theCcTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under theControl of the CcENO1 Promoter and the CcTEF1 Terminator

Plasmid pKK67 was digested with BamHI and XmnI. A 2442 bp fragment wasgel isolated and ligated to a 6031 bp fragment obtained by digesting aplasmid pKK77 with BamHI. The plasmid pKK67 contains the E. coli G418resistance gene under the control of the CcTEF1 promoter and the CcTPI1terminator and the plasmid pKK77 contains S. cerevisiae ALD6 encodinggene under the control of the CcENO1 promoter and the CcTEF1 terminator.The resulting plasmid was designated as pKK82 (FIG. 4).

Example 4B. Generation of a Genetically Modified C. curvatus (Y23/82)with an Integrated ALD6 Encoding Gene and G418 Resistance Gene byTransforming Wild-type C. curvatus with Digested Plasmid pKK82 (FIG. 4,Ex. 4A)

Plasmid pKK82 was restricted with NotI and PspOMI, and the resultinglinear DNA was used to transform wild-type C. curvatus strain ATCC 20509designated as Y23 by electroporation. The transformed cells werescreened for G418 resistance. Several G418-resistant colonies wereanalysed at DNA level by PCR. The transformants originating from thetransformation of C. curvatus with NotI and PspOM1 cut pKK82 andcontaining S. cerevisiae ALD6 encoding gene under the control of theCcENO1 promoter and the CcTEF1 terminator were designated as Y23/82-1,Y23/82-2, Y23/82-4 and Y23/82-13.

Example 5A. Construction of a Plasmid (pKK86, FIG. 5) Containing theHygromycin Resistance Gene Under the Control of the CcTEF1 Promoter andthe CcTPI1 Terminator and the S. cerevisiae ACS2 Encoding Gene Under theControl of the CcTPI1 Promoter and the CcENO1 Terminator

Plasmid pKK63 was digested with BamHI and SbfI. A 851 bp fragment wasgel isolated and ligated to a 3295 bp fragment obtained by digestingplasmid pKK60 with Ban-II and SbfI. The plasmid pKK63 contains theCcTPI1 promoter and the plasmid pKK60 contains the CcENO1 terminator.The resulting plasmid was designated as pKK78pre.

The plasmid UmACS contains the S. cerevisiae ACS2 (SEQ ID NO:50)encoding gene which has been codon optimized according to Ustilagomaydis yeast codon usage (SEQ ID NO:51) with flanking SbfI restrictionsites. Plasmid UmACS was digested with SbfI and DraI. A 2060 bp fragmentwas gel isolated and ligated to a 4146 bp fragment obtained by digestingplasmid pKK78pre with SbfI. The resulting plasmid was designated aspKK78.

Plasmid pKK76 was digested with BamHI and DraI. A 2652 bp fragment wasgel isolated and ligated to a 6206 bp fragment obtained by digestingplasmid pKK78 with BamHI. The plasmid pKK76 contains the E. colihygromycin resistance gene under the control of the CcTEF1 promoter andthe CcTPI1 terminator. The resulting plasmid was designated as pKK86(FIG. 5).

Example 5B. Generation of a Genetically Modified C. curvatus (Y23/86)with an Integrated ACS2 Encoding Gene and Hygromycin Resistance Gene byTransforming Wild-Type C. curvatus with Digested Plasmid pKK86 (FIG. 5,Ex. 5A)

Plasmid pKK86 was restricted with NotI and PspOMI, and the resultinglinear DNA was used to transform a wild-type C. curvatus strain ATCC20509 designated as Y23 by electroporation. The transformed cells werescreened for hygromycin resistance. Several hygromycin-resistantcolonies were analysed at DNA level by PCR. The transformantsoriginating from the transformation of C. curvatus with Moil and PspOMIcut pKK86 and containing S. cerevisiae ACS2 encoding gene under thecontrol of the CcTPI1 promoter and the CcENO1 terminator were designatedas Y23/86-86, Y23/86-92, Y23/86-93, Y23/86-98 and Y23/86-100.

Example 6A. Construction of a Plasmid (pKK95, FIG. 6) Containing theCerulenin Resistance Gene Under the Control of the CcTEF1 Promoter andthe CcTPI1 Terminator and the R. oryzae PDAT Encoding Gene Under theControl of the CcTPI1 Promoter and the CcGPD1 Terminator

Plasmid pKK54 was digested with KpnI and SbfI. A 394 bp fragment was gelisolated and ligated to a 3742 bp fragment obtained by digesting plasmiddesignated as pKK63 with KpnI and SOL The plasmid pKK54 contains theCcGPD1 terminator and the plasmid pKK63 contains the CcTPI1 promoter.The resulting plasmid was designated as pKK93pre. Plasmid UmPDAT wasdigested with SbfI. A 1844 bp fragment was gel isolated and ligated to a4136 bp fragment obtained by digesting plasmid pKK93pre with SbfI. Theplasmid UmPDAT contains a R. oryzae PDAT (SEQ ID NO:52) encoding genewhich has been codon optimized according to U. maydis yeast codon usage(SEQ ID NO:53) with flanking SbfI restriction sites. The resultingplasmid was designated as pKK93.

The plasmid pKK93 was digested with EcoRI and DraI. A 3029 bp fragmentwas gel isolated and ligated to a 6242 bp fragment obtained by digestingplasmid pKK91 with EcoRI. The plasmid pKK91 contains the S. cerevisiaecerulenin resistance gene under the control of the CcTEF1 promoter andthe CcTPI1 terminator. The resulting plasmid was designated as pKK95(FIG. 6).

Example 6B. Generation of a Genetically Modified C. curvatus (Y23/95)with an Integrated PDAT Encoding Gene and Cerulenin Resistance Gene byTransforming Wild-Type C. curvatus with Digested Plasmid pKK95 (FIG. 6,Ex. 6A)

Plasmid pKK95 was restricted with EcoRV, and the resulting linear DNAwas used to transform a wild-type C. curvatus strain ATCC 20509designated as Y23 by electroporation. The transformed cells werescreened for cerulenin resistance. Several cerulenin-resistant colonieswere analysed at DNA level by PCR. The transformants originating fromthe transformation of C. curvatus with EcoRV cut pKK95 and containingthe R. oryzae PDAT encoding gene under the control of the CcTPI1promoter and the CcGPD1 terminator were designated as Y23/95-87,Y23/95-98, Y23/95-99, Y23/95-104 and Y23/95-109.

Example 7A. Construction of a Plasmid (pKK85, FIG. 7) Containing theHygromycin Resistance Gene Under the Control of the CcTEF1 Promoter andthe CcTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under theControl of the CcENO1 Promoter and the CcTEF1 Terminator and the S.cerevisiae ACS2 Encoding Gene Under the Control of the CcTPI1 Promoterand the CcENO1 Terminator

Plasmid pKK77 was digested with EcoRI and XmnI. A 3073 bp fragment wasgel isolated and ligated to a 6206 bp fragment obtained by digestingplasmid pKK78 with EcoRI. The plasmid pKK77 contains the S. cerevisiaeALD6 encoding gene under the control of the CcENO1 promoter and theCcTEF1 terminator and the plasmid pKK78 contains the S. cerevisiae ACS2encoding gene under the control of the CcTPI1 promoter and the CcENO1terminator. The resulting plasmid was designated as pKK84. The plasmidpKK76 was digested with BamHI and XmnI. A 2652 bp fragment was gelisolated and ligated to a 9279 bp fragment obtained by digesting pKK84with BamHI. The plasmid pKK76 contains the hygromycin resistance geneunder the control of the CcTEF1 promoter and the CcTPI1 terminator. Theresulting plasmid was designated as pKK85 (FIG. 7).

Example 7B. Generation of a Genetically Modified C. curvatus (Y23/85)with an Integrated ALD6 Encoding and ACS2 Encoding Genes and HygromycinResistance Gene by Transforming Wild-Type C. curvatus with DigestedPlasmid pKK85 (FIG. 7, Ex. 7A)

Plasmid pKK85 was restricted with NotI and PspOMI, and the resultinglinear DNA was used to transform wild-type C. curvatus strain ATCC 20509designated as Y23 by electroporation. The transformed cells werescreened for hygromycin resistance. Several hygromycin-resistantcolonies were analysed at DNA level by PCR. The transformantsoriginating from the transformation of C. curvatus with NotI and PspOMIcut pKK85 and containing the S. cerevisiae ALD6 encoding gene under thecontrol of the CcENO1 promoter and the CcTEF1 terminator and the S.cerevisiae ACS2 encoding gene under the control of the CcTPI1 promoterand the CcENO1 terminator were designated as Y23/85-119, Y23/85-125,Y23/85-128 Y23/85-129 and Y23/85-139.

Example 8. Generation of a Genetically Modified C. curvatus (Y23/81/95)with an Integrated ALD6 Encoding and PDAT Encoding Genes and Hygromycinand Cerulenin Resistance Genes by Transforming Genetically ModifiedStrain Y23/81-51 (Ex. 3B) with Plasmid pKK95 (FIG. 6, Ex. 6A)

Plasmid pKK95 was restricted with EcoRV and the resulting linear DNA wasused to transform a genetically modified strain Y23/81-51 byelectroporation. The transformed cells were screened for cerulenin andhygromycin resistance. Several cerulenin and hygromycin resistancecolonies were analysed at DNA level by PCR. The transformantsoriginating from the transformation of genetically modified strainY23/81-51 with EcoRV cut pKK95 and containing the S. cerevisiae ALD6encoding gene under the control of the CcENO1 promoter and the CcTEF1terminator and the R. oryzae PDAT encoding gene under the control of theCcTPI1 promoter and the CcGPD1 terminator were designated asY23/81/95-18 and Y23/81/95-42.

Example 9. Generation of a Genetically Modified C. curvatus (Y23/85/95)with an Integrated ALD6, ACS2 and PDAT Encoding Genes and Hygromycin andCerulenin Resistance Genes by Transforming Genetically Modified StrainY23/85-128 (Ex. 7B) with Plasmid pKK95 (FIG. 6, Ex. 6A)

Plasmid pKK95 was restricted with EcoRV and the resulting linear DNA wasused to transform a genetically modified strain Y23/85-128 byelectroporation. The transformed cells were screened for cerulenin andhygromycin resistance. Several cerulenin and hygromycin resistancecolonies were analysed at DNA level by PCR. The transformantsoriginating from the transformation of genetically modified strainY23/85-128 with EcoRV cut pKK95 and containing the S. cerevisiae ALD6encoding gene under the control of the CcENO1 promoter and the CcTEF1terminator, the S. cerevisiae ACS2 encoding gene under the control ofthe CcTPI1 promoter and the CcENO1 terminator and R. oryzae PDATencoding gene under the control of the CcTPI1 promoter and the CcGPD1terminator were designated as Y23/85/95-4 and Y23/85/95-68.

Example 10. Cloning of Mucor circinelloides TPI (McTPI1) Promoter andTerminator Region

A genomic fragment of the M. circinelloides TPI1 gene was amplified byPCR from genomic M. circinelloides f. griseocyanus (D-82202, VTT CultureCollection) DNA with degenerative primers identified as SEQ ID NO:54(Mould TPI1) and SEQ ID NO:55 (Mould TPI3). The degenerative primerswere designed based on a consensus sequence of TPI1 genes of Rhizopusoryzae, Fusarium oxysporum, Aspergillus fumigatus, A. terreus and A.nidulans. A ˜400 bp genomic fragment was isolated and sequenced.

A genomic fragment containing the McTPI1 promoter region was obtainedwith a ligation-mediated PCR amplification with TPI1 gene specificoligonucleotides identified as SEQ ID NO:56 (MC_TPI2) and SEQ ID NO:57(MC_TPI1), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that SspI-digested M. circinelloides DNA was used. A ˜1500 bp PCRfragment was isolated and sequenced.

The M. circinelloides TPI1 promoter was PCR amplified by using primersidentified as SEQ ID NO:58 (MC_TPI1) and SEQ ID NO:59 (MC_TPI_8), andthe M. circinelloides genomic DNA as the template. A PCR fragment wasdigested with PstI and BamHI. A 1251 bp fragment was gel isolated andligated to a PstI and BamHI-digested pBluescript KS-plasmid. Theresulting plasmid was designated pKK56. Plasmid pKK56 contains the M.circinelloides TPI1 promoter.

A genomic fragment containing the McTPI1 terminator region was obtainedwith a ligation-mediated PCR amplification with TPI1 gene specificoligonucleotides identified as SEQ ID NO:60 (MC_TPI4) and SEQ ID NO:61(MC_TPI3), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that NruI-digested M. circinelloides DNA was used. A ˜1500 bp PCRfragment was isolated and partially sequenced.

The M. circinelloides TPI1 terminator was PCR amplified by using primersidentified as SEQ ID NO:62 (MC_TPI5) and SEQ ID NO:63 (MC_TPI6), and theM. circinelloides genomic DNA as the template. A PCR fragment wasdigested with XbaI and BamHI. A 347 bp fragment was gel isolated andligated to a XbaI and BamHI-digested pBluescript KS-plasmid. Theresulting plasmid was designated pKK57. Plasmid pKK57 contains the M.circinelloides TPI1 terminator.

Example 11. Cloning of Mucor circinelloides TEF (McTEF1) Promoter andTerminator Region

A genomic fragment of the M. circinelloides TEF1 gene was amplified byPCR from genomic M. circinelloides f. griseocyanus (D-82202, VTT CultureCollection) DNA with degenerative primers identified as SEQ ID NO:64(Mould TEF1) and SEQ ID NO:65 (Mould TEF4). The degenerative primerswere designed based on a consensus sequence of TEF1 genes of Rhizopusoryzae, Fusarium oxysporum, Aspergillus terreus and A. nidulans. A ˜600bp genomic fragment was isolated and sequenced.

A genomic fragment containing the McTEF1 promoter region was obtainedwith a ligation-mediated PCR amplification with TEF1 gene specificoligonucleotides identified as SEQ ID NO:66 (MC_TEF2) and SEQ ID NO:67(MC_TEF1), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that SspI-digested M. circinelloides DNA was used. A ˜500 bp PCRfragment was isolated and sequenced. Nested primers identified as SEQ IDNO:68 (MC_TEF6) and SEQ ID NO:69 (MC_TEF5) were designed and used in aligation-mediated PCR amplification together with oligonucleotidesidentified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR LinkerII), similarly as above except that HaeIII-digested M. circinelloidesDNA was used. A ˜1500 bp PCR fragment was isolated and sequenced.

The M. circinelloides TEF1 promoter was PCR amplified by using primersidentified as SEQ ID NO:70 (MC_TEF9) and SEQ ID NO:71 (MC_TEF10), andthe M. circinelloides genomic DNA as the template. A PCR fragment wasdigested with HindIII and PstI and a 1387 bp fragment was gel isolatedand ligated to a HindIII and PstI-digested pBluescript KS-plasmid. Theresulting plasmid was designated pKK64. Plasmid pKK64 contains the M.circinelloides TEF1 promoter.

A genomic fragment containing the McTEF1 terminator region was obtainedwith a ligation-mediated PCR amplification with TPI1 gene specificoligonucleotides identified as SEQ ID NO:72 (MC_TEF4) and SEQ ID NO:73(MC_TEF3), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that SspI-digested M. circinelloides DNA was used. A ˜1100 bp PCRfragment was isolated and sequenced. Nested primers identified as SEQ IDNO:74 (MC_TEF8) and SEQ ID NO:75 (MC_TEF7) were designed and used in aligation-mediated PCR amplification together with oligonucleotidesidentified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR LinkerII), similarly as above except that HpaI-digested M. circinelloides DNAwas used. A ˜1200 bp PCR fragment was isolated and sequenced.

The M. circinelloides TEF1 terminator was PCR amplified by using primersidentified as SEQ ID NO:76 (MC_TEF11) and SEQ ID NO:77 (MC_TEF12), andthe M. circinelloides genomic DNA as the template. A PCR fragment wasdigested with XmaI and EcoRI and a 389 bp fragment was gel isolated andligated to a XmaI and EcoRI-digested pBluescript KS-plasmid. Theresulting plasmid was designated pKK65. Plasmid pKK65 contains the M.circinelloides TEF1 terminator.

Example 12. Cloning of Mucor circinelloides PGK (McPGK1) Promoter Region

A genomic fragment of the M. circinelloides PGK1 gene was amplified byPCR from genomic M. circinelloides griseocyanus (D-82202, VTT CultureCollection) DNA with degenerative primers identified as SEQ ID NO:78(Mould PGK4) and SEQ ID NO:79 (Mould PGK2). The degenerative primerswere designed based on a consensus sequence of PGK1 genes of Rhizopusoryzae, Fusarium oxysporum, Aspergillus fumigatus, A. oryzae and A.nidulans. A ˜250 bp genomic fragment was isolated and sequenced.

A genomic fragment containing the McPGK1 promoter region was obtainedwith a ligation-mediated PCR amplification with PGK1 gene specificoligonucleotides identified as SEQ ID NO:80 (MC_PGK2) and SEQ ID NO:81(MC_PGK1), together with oligonucleotides identified as SEQ ID NO:3 (PCRLinker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1Aexcept that SspI-digested M. circinelloides DNA was used. A ˜1300 bp PCRfragment was isolated and sequenced. Nested primers identified as SEQ IDNO:82 (MC_PGK4) and SEQ ID NO:83 (MC_PGK3) were designed and used in aligation-mediated PCR amplification together with oligonucleotidesidentified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR LinkerII), similarly as above except that HaeIII-digested M. circinelloidesDNA was used. A ˜600 bp PCR fragment was isolated and sequenced.

The M. circinelloides PGK1 promoter was PCR amplified by using primersidentified as SEQ ID NO:84 (MC_PGK5) and SEQ ID NO:85 (MC_PGK6), and theM. circinelloides genomic DNA as the template. A PCR fragment wasdigested with SacII and XbaI and a 1291 bp fragment was gel isolated andligated to a SacII and XbaI-digested pBluescript KS-plasmid. Theresulting plasmid was designated pKK62. Plasmid pKK62 contains the M.circinelloides PGK1 promoter.

Example 13. Cloning of Mucor circinelloides GPD (McGPD1) Promoter Region

A genomic fragment containing the McGPD1 promoter region was obtainedwith a ligation-mediated PCR amplification with Mucor circinelloides(Syn. racemosus) GPD1 gene (GenBank accession number AJ293012, versionnumber AJ293012.1) specific oligonucleotides identified as SEQ ID NO:86(MC_GPD2) and SEQ ID NO:87 (MC_GPD1), together with oligonucleotidesidentified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR LinkerII), similarly as in Example 1A except that EcoRV-digested M.circinelloides (D-82202, VTT Culture Collection) DNA was used. A ˜500 bpPCR fragment was isolated and sequenced. Nested primers identified asSEQ ID NO:88 (MC_GPD10) and SEQ ID NO:89 (MC_GPD9) were designed andused in a ligation-mediated PCR amplification together witholigonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ IDNO:4 (PCR Linker II), similarly as above except that StuI-digested M.circinelloides DNA was used. A ˜1400 bp PCR fragment was isolated andsequenced.

The M. circinelloides GPD1 promoter was PCR amplified by using primersidentified as SEQ ID NO:90 (MC_GPD11) and SEQ ID NO:91 (MC_GPD12), andthe M. circinelloides genomic DNA as the template. A PCR fragment wasdigested with EcoRI and HindIII and a 1440 bp fragment was gel isolatedand ligated to a EcoRI and HindIII-digested pBluescript KS-plasmid. Theresulting plasmid was designated pKK59. Plasmid pKK59 contains the M.circinelloides GPD1 promoter.

Example 14A: Cloning of E. coli Hygromycin Resistance Gene; Constructionof a Plasmid (pKK69) Having the E. coli Hygromycin Resistance Gene Underthe Control of the McPGK1 Promoter and the McTPI1 Terminator

Plasmid pKK62 was digested with SacII and SbfI. A 1286 bp fragment wasgel isolated. Plasmid pKK52 was digested with SbfI. A 1034 bp fragmentwas gel isolated. The 1286 bp fragment originated from plasmid pKK62 andthe 1034 bp fragment originated from plasmid pKK52 were ligated to a3268 bp fragment obtained by digesting plasmid pKK57 with SacII and W.Plasmid pKK52 contains the E. coli hygromycin resistance gene, plasmidpKK62 contains the M. circinelloides PGK1 promoter and plasmid pKK57contains the M. circinelloides TPI1 terminator. The resulting plasmidwas designated pKK69. Plasmid pKK69 contains the E. coli hygromycinresistance gene under the control of the M. circinelloides PGK1 promoterand the M. circinelloides TPI1 terminator.

Example 14B: Cloning of S. cerevisiae Cerulenin Resistance Gene;Construction of a Plasmid (pKK92) Having the S. cerevisiae CeruleninResistance Gene Under the Control of the McPGK1 Promoter and the McTPI1Terminator

Plasmid pKK80 was digested with PstI. A 1685 bp fragment was gelisolated. Plasmid pKK80 contains the S. cerevisiae cerulenin resistancegene. The 1685 bp fragment was ligated to a 4554 bp fragment obtained bydigesting plasmid pKK69 with SbfI. Plasmid pKK69 contains the M.circinelloides PGK1 promoter and the M circinelloides TRI1 terminator.The resulting plasmid was designated pKK92. Plasmid pKK92 contains theS. cerevisiae cerulenin resistance gene under the control of the M.circinelloides PGK1 promoter and the M. circinelloides TPI1 terminator.

Example 15A. Construction of a Plasmid (pKK75, FIG. 8) Containing theHygromycin Resistance Gene Under the Control of the McPGK1 Promoter andthe McTPI1 Terminator and the S. cerevisiae ALD6 Gene Under the Controlof the McTPI1 Promoter and the McTEF1 Terminator

Plasmid pKK56 was digested with BamHI and PstI. A 1251 bp fragment wasgel isolated. Plasmid pKK56 contains the McTPI1 promoter. Plasmid RoALDwas digested with SbfI. A 1514 bp fragment was gel isolated. The plasmidRoALD contains the S. cerevisiae ALD6 (SEQ ID NO:47) encoding gene whichhas been codon optimized according to Rhizopus oryzae filamentous funguscodon usage (SEQ ID NO:49) with flanking SbfI restriction sites. The1251 bp fragment originating from the plasmid pKK56 and the 1514 bpfragment originating from the plasmid RoALD were ligated to a 3321 bpfragment obtained by digesting plasmid pKK65 with BamHI and SbfI.Plasmid pKK65 contains the McTEF1 terminator. The resulting plasmid wasdesignated as pKK73. Plasmid pKK73 contains the S. cerevisiae ALD6encoding gene under the control of the M. circinelloides TPI1 promoterand the M. circinelloides TEF1 terminator.

Plasmid pKK69 was digested with BamHI and XmnI. A 2652 bp fragment wasgel isolated and ligated to a 6086 bp fragment obtained by digestingplasmid pKK73 with BamHI. Plasmid pKK69 contains the E. coli hygromycinresistance gene under the control of the M. circinelloides PGK1 promoterand the M. circinelloides TPI1 terminator. The resulting plasmid wasdesignated as pKK75 (FIG. 8).

Example 15B. Generation of a Genetically Modified Mucor circinelloides(M22/75) with an Integrated ALD6 Encoding Gene and a HygromycinResistance Gene by Transforming Wild-Type M. circinelloides withDigested Plasmid pKK75 (FIG. 8, Ex. 15A)

Plasmid pKK75 was restricted with KpnI and NotI. A 5866 bp fragment wasgel isolated and used to transform a wild-type M. circinelloides strain(D-82202, VTT Culture Collection) designated as M22, using a Mucorprotoplast transformation method (Wolff et. al. 2002). The transformedcells were screened for hygromycin resistance. Severalhygromycin-resistant colonies were analysed at DNA level by PCR. Thetransformants originating from the transformation of the wild-type M.circinelloides strain with KpnI and NotI cut pKK75 and containing the S.cerevisiae ALD6 encoding gene under the control of the McTPI1 promoterand the McTEF1 terminator were designated as M22/75-80 and M22/75-86.

Example 16A. Construction of a Plasmid (pKK94, FIG. 9) Containing theHygromycin Resistance Gene Under the Control of the McPGK1 Promoter andthe McTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under theControl of the McTPI1 Promoter and the McTEF1 Terminator and the S.cerevisiae ACS2 Encoding Gene Under the Control of the McTEF1 Promoterand the McTPI1 Terminator

Plasmid pKK57 was digested with PstI. A 351 bp fragment was gel isolatedand ligated to a 4326 bp fragment obtained by digesting plasmid pKK64with PstI. The plasmid pKK57 contains the McTPI terminator and theplasmid pKK64 contains the McTEF promoter. The resulting plasmid wasdesignated pKK90Pre. Plasmid RoACS was digested with SbfI. A 2060 bpfragment was gel isolated and ligated to a 4677 bp fragment obtained bydigesting plasmid pKK90Pre with SbfI. The plasmid RoACS contains the S.cerevisiae ACS2 (SEQ ID NO:50) encoding gene which has been codonoptimized according to Rhizopus oryzae filamentous fungus codon usage(SEQ ID NO:92) with flanking SbfI restriction sites. The resultingplasmid was designated as pKK90.

Plasmid pKK75 was digested with KpnI followed by removal of the 3′overhangs by T4 DNA polymerase and each of the 4 dNTPs. KpnI(blunt)-digested plasmid pKK75 was digested with NotI and a 5866 bpfragment was gel isolated. The plasmid pKK75 contains the E. colihygromycin resistance gene under the control of the M. circinelloidesPGK1 promoter and the M. circinelloides TPI1 terminator and S.cerevisiae ALD6 encoding gene under the control of the M. circinelloidesTPI1 promotor and the M. circinelloides TEF1 terminator. The 5866 bpfragment originating from plasmid pKK75 was ligated to a 6698 bpfragment obtained by digesting plasmid pKK90 with SmaI and NotI. Theresulting plasmid was designated as pKK94 (FIG. 9).

Example 16B. Generation of a Genetically Modified Mucor circinelloides(M22/94) with an Integrated ALD6 and ACS2 Encoding Genes and aHygromycin Resistance Gene by Transforming Wild-Type M. circinelloideswith Digested Plasmid pKK94 (FIG. 9, Ex. 16A)

Plasmid pKK94 was restricted with KpnI and SacI. A 9701 bp fragment wasgel isolated and used to transform a wild-type M. circinelloides strain(D-82202, VTT Culture Collection) designated as M22, using thetransformation method described in Example 15B. The transformed cellswere screened for hygromycin resistance. Several hygromycin resistantcolonies were analysed at DNA level by PCR. The transformantsoriginating from the transformation of a wild-type M. circinelloidesstrain with KpnI and SacI cut pKK94 and containing the S. cerevisiaeALD6 encoding gene under the control of the McTPI1 promoter and theMcTEF1 terminator and S. cerevisiae ACS2 encoding gene under the controlof the McTEF1 promoter and the McTPI1 terminator were designated asM22/94-12, M22/94-16 and M22/94-24.

Example 17A. Construction of a Plasmid (pKK96, FIG. 10) Containing theHygromycin Gene Under the Control of the McPGK1 Promoter and the McTPI1Terminator and the S. cerevisiae ACS2 Encoding Gene Under the Control ofthe McTEF1 Promoter and the McTPI1 Terminator

Plasmid pKK94 was digested with BsrGI and a 9683 bp fragment was gelisolated and self ligated. The resulting plasmid was designated as pKK96(FIG. 10). The plasmid pKK96D contains the S. cerevisiae ACS2 encodinggene under the control of the McTEF1 promoter and the McTPI1 terminator.

Example 17B. Generation of a Genetically Modified Mucor circinelloides(M22/96) with an Integrated ACS2 Encoding Gene and Hygromycin ResistanceGene by Transforming Wild-Type M. circinelloides with Digested PlasmidpKK96 (FIG. 10, Ex. 17A)

Plasmid pKK96 was restricted with KpnI and SacI. A 6824 bp fragment wasgel isolated and used to transform the wild-type M. circinelloidesstrain (D-82202, VTT Culture Collection) designated as M22, using thetransformation method described in Example 15B. The transformed cellswere screened for hygromycin resistance. Several hygromycin resistantcolonies were analysed at DNA level by PCR. The transformantsoriginating from the transformation of the wild-type M. circinelloidesstrain with KpnI and SacI cut pKK96D and containing the S. cerevisiaeACS2 encoding gene under the control of the McTEF1 promoter and theMcTPI1 terminator were designated as M22/96-1 and M22/96-6.

Example 18A. Construction of a Plasmid (pKK98) Containing the CeruleninResistance Gene Under the Control of the McPGK1 Promoter and the McTPI1Terminator and the R. oryzae PDAT Gene Under the Control of the McGPD1Promoter and the McTEF1 Terminator

Plasmid pKK59 was digested with SbfI and XbaI. A 1467 bp fragment wasgel isolated and ligated to a 3309 bp fragment obtained by digestingplasmid pKK65 with SbfI and XbaI. The plasmid pKK59 contains the McGPD1promoter and the plasmid pKK65 contains the McTEF1 terminator. Theresulting plasmid was designated as pKK88. Plasmid RoPDAT was digestedwith SbfI. A 1844 bp fragment was gel isolated and ligated to a 4776 bpfragment obtained by digesting plasmid pKK88 with SbfI. The plasmidRoPDAT contains the R. oryzae PDAT (SEQ ID NO:52) encoding gene, whichhas been codon optimized according to R. oryzae codon usage (SEQ. ID. NO93) with flanking SbfI restriction sites. The resulting plasmid wasdesignated as pKK97.

Plasmid pKK97 was digested with EcoRI. A 3659 bp fragment was gelisolated and ligated to a 6239 bp fragment obtained by digesting plasmidpKK92 with EcoRI. The plasmid pKK92 contains the S. cerevisiae ceruleninresistance gene under the control of the McPGK1 promoter and the McTPI1terminator. The resulting plasmid was designated as pKK98 (FIG. 11).

Example 18B. Generation of a Genetically Modified Mucor circinelloides(M22/98) with an Integrated PDAT Encoding Gene and Cerulenin ResistanceGene by Transforming Wild-Type M. circinelloides with Digested PlasmidpKK98 (FIG. 11, Ex. 18A)

Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp fragment wasgel isolated and used to transform the wild-type M. circinelloidesstrain (D-82202, VTT Culture Collection) designated as M22, using thetransformation method described in Example 15B. The transformed cellswere screened for cerulenin resistance. Several cerulenin resistantcolonies were analysed at DNA level by PCR. The transformantsoriginating from the transformation of the wild-type M. circinelloidesstrain with ApaI and SacII cut pKK98 and containing the R. oryzae PDATencoding gene under the control of the McGPD1 promoter and the McTEF1terminator were designated as M22/98-16.

Example 19. Generation of a Genetically Modified Mucor circinelloides(M22/75/98) with Integrated ALD6 and PDAT Encoding Genes and Hygromycinand Cerulenin Resistance Genes by Transforming Genetically ModifiedStrain M22/75-86 (Ex 15B) with Digested Plasmid pKK98 (FIG. 11, Ex. 18A)

Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp fragment wasgel isolated and used to transform the genetically modified strainM22/75-86, using the transformation method described in Example 15B. Thetransformed cells were screened for cerulenin resistance. Severalcerulenin resistant colonies were analysed at DNA level by PCR. Thetransformants originating from the transformation of the recombinantM22/75-86 strain with ApaI and SacII cut pKK98 and containing the R.oryzae PDAT encoding gene under the control of the McGPD1 promoter andthe McTEF1 terminator and the S. cerevisiae ALD6 encoding gene under thecontrol of the McTPI1 promoter and the McTEF1 terminator were designatedas M22/75/98-7 and M22/75/98-9.

Example 20. Generation of a Genetically Modified Mucor circinelloides(M22/94/98) with Integrated ALD6, ACS2 and PDAT Encoding Genes andHygromycin and Cerulenin Resistance Genes by Transforming GeneticallyModified Strain M22/94-31 (Ex 16B) with Digested Plasmid pKK98 (FIG. 11,Ex. 18A)

Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp fragment wasgel isolated and used to transform the genetically modified strainM22/94-31, using the transformation method described in Example 15B. Thetransformed cells were screened for cerulenin resistance. Severalcerulenin resistant colonies were analysed at DNA level by PCR. Thetransformants originating from the transformation of the recombinantM22/94-31 strain with ApaI and SacII cut pKK98 and containing the R.oryzae PDAT encoding gene under the control of the McGPD1 promoter andthe McTEF1 terminator, the S. cerevisiae ALD6 encoding gene under thecontrol of the McTPI1 promoter and the McTEF1 terminator and the S.cerevisiae ACS2 encoding gene under the control of the McTEF1 promoterand the McTPI1 terminator were designated as M22/94/98-19 andM22/94/98-22.

Example 21. Lipid Extraction and Total Lipid and TriglycerideConcentration Measurements

A lipid extraction method was modified from the protocol of Folch etal., 1957. 0.5 to 2 ml of cell culture was taken into an Eppendorf tube.The sample was centrifuged and the supernatant discarded. The pellet wasplaced rapidly in liquid nitrogen and stored at −80° C. Alternatively,filamentous fungal cells of 2 to 12 ml culture broth were collected byvacuum filtration through disks of glass microfiber filters (Whatman,England). After washing twice with distilled water, biomass was removedfrom the filter using a clean spatula and put into 2 ml microfuge tubes,which were placed rapidly in liquid nitrogen and stored at −80° C. In ahomogenisation step the frozen pellet was suspended in 500 μl ofice-cold methanol with 0.1% BHT (2,6-Di-tertbutyl-4-methylphenol) andhomogenised with a Mixer Mill homogenizator with 5-mm zirconium oxideand 3-mm yttrium stabilized zirconium oxide balls (Retsch) at 25 Hz for5 min. After homogenisation 1000 μl of chloroform was added andhomogenisation repeated. After re-homogenisation 300 μl of 20 mM aceticacid was added and the sample vortexed for 10 min. After vortexing thesample was centrifuged 13 000 rpm for 5 min at RT. The lower phase wasrecovered and 1000 μl of chloroform was added to the remaining phase,vortexed and recentrifuged. The lower phases were combined intopre-weighed 2 ml microfuge tubes, and dried, after which the total lipidcontent of the sample was determined by gravimetry. Then the lipidsample was redissolved in 1.5 ml of chloroform:methanol (2:1)+0.1% BHTand stored at −20° C. For triacylglycerol analysis 100 to 1500 μl ofchloroform:methanol extracted lipids were evaporated and re-dissolved in200-1000 μl of isopropanol. Triacylglycerols were measured enzymaticallyfrom the samples by using the Konelab Triglycerides Kit (ThermoScientific, Finland) and Cobas Mira automated analyser (Roche) or amicrotitre-plate reader (Varioskan, Thermo Electron Corporation). Thislipid extraction and total lipid and triglyceride concentrationmeasurements methods were used in the following examples if nototherwise indicated.

Example 22. Microaerobic Shake Flask Characterization of StrainsY23/81-51 and Y23/81-66 (Ex. 3B), Y23/86-86 and Y23/86-92 (Ex. 5B),Y23/95-87 and Y23/95-109 (Ex. 6B) and Y23/85/95-4 (Ex. 9), in GlucoseMedium with C/N Ratio of 20

Transformants were separately cultivated in 50 ml of culture medium“glucose CN20” (pH 5.5, 20 g glucose, 0.3 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5g Na₂HPO₄*2H₂O, 1.5 g MgSO₄*7H₂O, 4.0 g Yeast extract, 51 mg CaCl₂, 8 mgFeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) wasinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 100 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis were withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 was used as acontrol.

Lipid extraction and triacylglycerol concentration measurement werecarried out as described in Example 21. Cell dry weight was determinedby centrifuging 1 ml of the culture broth in pre-dried, pre-weighedEppendorf tubes. After washing with 1 ml of distilled water the cellpellet was dried at 100° C. for 24 hours and weighed again. HPLCanalyses for sugars were conducted with a Waters 2690 Separation Moduleand Water System Interfase Module liquid chromatography coupled with aWaters 2414 differential refractometer and Waters 2487 dual absorbancedetector. The liquid chromatography columns were a 100×7.8 mm Fast AcidAnalysis column from Bio-Rad and a 300×7.8 mm Aminex HPX-87H column fromBio-Rad. The columns were equilibrated with 2.5 mM H₂SO₄ in water at 55°C. and samples were eluted with 2.5 mM H₂SO₄ in water at 0.5 ml/min flowrate. Data acquisition was done using Waters Millennium software. ThisHPLC method was used in all appropriate Examples.

After 48 hours cultivation (Table 2A), when 4 to 8 g/l glucose was left,Y23/81, Y23/86, Y23/95 and Y23/85/95 transformants produced 12, 22, 15and 24% more triacylglycerols with higher rate, respectively, than thecontrol strain in glucose medium. Triacylglycerol yields per usedglucose were also better up to 10% with the transformants compared tothe control strain. Additionally Y23/85/95 transformant had 13% highertriacylglycerol yield on biomass than the control strain (12.9 and 11.4%TAG yield on biomass, respectively). In particular, after 24 hours ofcultivation (Table 2B), the strains expressing ALD or ACS alone, or ALDand ACS together, had enhanced production of triacylglycerols, measuredas concentration (g/l) and as yield per biomass and used glucose, withhigher rate (mg/l/h) compared to the control strain.

TABLE 2A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) andyield (%) per used glucose after 48 hours microaerobic cultivation inglucose medium with C/N ratio of 20 Yield TAG TAG (% used TAG Strain(g/l) glucose) mg/l/h Control 0.72 5.88 15.0 Y23/81 (ALD) 0.81 5.90 16.8Y23/86 (ACS) 0.88 6.00 18.3 Y23/95 (PDAT) 0.82 6.02 17.2 Y23/85/95 0.896.46 18.5 (ALD + ACS + PDAT)

TABLE 2B Triacylglycerol (TAG) concentration (g/l) and yield (%) perbiomass (CDW) and used glucose after 24 hours microaerobic cultivationin glucose medium with C/N ratio of 20 TAG Yield TAG Yield TAG TAGStrain (g/l) (% CDW) (% used glucose) mg/l/h Control 0.22 5.15 4.29 9.1Y23/81-66 (ALD) 0.26 6.09 4.86 10.8 Y23/85-125 (ALD + 0.31 7.01 5.4912.8 ACS) Y23/86-86 (ACS) 0.30 6.76 5.26 12.5

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations and ratesof production, and triacylglycerol yields from used glucose or per dryweight in cultivation with low C/N ratios.

Example 23. Aerobic Shake Flask Characterization of Strains Y23/81-8,51, 59, 66 and 69 (Ex. 3B), Y23/85-119, 125, 128, 129 and 139 (Ex. 7B),Y23/86-86, 92, 93, 98 and 100 (Ex. 5B), Y23/95-99 and 104 (Ex. 6B),Y23/81/95-42 (Ex. 8) and Y23/85/95-4 and 68 (Ex. 9), in Glucose Mediumwith C/N Ratio of 65

Transformants were separately cultivated in 50 ml of Yeast culturemedium II (pH 5.5, 20 g glucose, 0.3 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5 gNa₂HPO₄*2H₂O, 1.5 g MgSO₄*7H₂O, 0.6 g Yeast extract, 51 mg CaCl₂, 8 mgFeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) wasinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 250 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis were withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 was used as acontrol. Cell dry weight was determined and HPLC analysis was carriedout as described in Example 22. Lipid extraction and triacylglycerolmeasurements as described in Example 21.

After 44 hours cultivation (Table 3A) transformants Y23/81, Y23/85,Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had 4, 3, 6, 1, 12 and 4% bettertriacylglycerol yields per used glucose, respectively, than the controlstrain. Additionally, transformants Y23/81/95 and Y23/85/95 had 7-8%higher triacylglycerol yield on biomass than the control strain (42.8,43.0 and 40.0% TAG yield [/CDW], respectively). After 25 hourscultivation (Table 3B), the strains expressing ALD or ACS alone, or ALDand ACS together, had enhanced production of triacylglycerols, measuredas concentration (g/l) and as yield per biomass and used glucose.

TABLE 3A Maximal triacylglycerol (TAG) yield (%) per used glucose after44 hours cultivation in glucose medium with C/N ratio of 65 Yield TAGStrain (% used glucose) Control 16.6 Y23/81 (ALD) 17.3 Y23/85 (ALD +ACS) 17.2 Y23/86 (ACS) 17.6 Y23/95 (PDAT) 16.9 Y23/81/95 (ALD + PDAT)18.6 Y23/85/95 (ALD + ACS + PDAT) 17.4

TABLE 3B Triacylglycerol (TAG) concentration (g/l) and yield (%) perbiomass (CDW) and used glucose after 25 hours cultivation in glucosemedium with C/N ratio of 65 TAG Yield TAG Yield TAG TAG Strain (g/l) (%CDW) (% used glucose) mg/l/h Control 1.01 21.7 10.3 40.5 Y23/81-51, 66(ALD) 1.15 26.4 12.5 46.0 Y23/85-125, 129 1.14 27.2 13.3 45.4 (ALD +ACS) Y23/86-92, 100 (ACS) 1.18 22.4 12.0 47.1

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations and ratesof production and triacylglycerol yields from used glucose or per dryweight.

Example 24. Aerobic Shake Flask Characterization of Strains Y23/81-66(Ex. 3B), Y23/85-125 (Ex. 7B), Y23/86-92 (Ex. 5B), Y23/95-98 (Ex. 6B)and Y23/81/95-18 (Ex. 8) in Glucose Medium with C/N Ratio of 103

Transformants were separately cultivated in 50 ml of Yeast culturemedium IV (pH 5.5, 20 g glucose, 0.15 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5 gNa₂HPO₄*2 H₂O, 1.5 g MgSO₄*7H₂O, 0.45 g Yeast extract, 51 mg CaCl₂, 8 mgFeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) wasinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 250 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis were withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 was used as acontrol. Cell dry weight was determined and HPLC analysis was carriedout as described in Example 22. Lipid extraction and triacylglycerolmeasurements as described in Example 21.

After 35 hours cultivation, when 6-8 g/l glucose was left, transformantsY23/81-66, Y23/85-125, Y23/86-92, Y23/95-98 and Y23/81/95-18 had 3, 6,9, 11 and 11% higher triacylglycerol titre and rate than the controlstrain, respectively. The transformants Y23/81-66, Y23/85-125,Y23/86-92, Y23/95-98 and Y23/81/95-18 had also 11, 19, 7, 20 and 30%better triacylglycerol yields per used glucose, respectively, than thecontrol strain. Additionally Y23/81/95-18 had 24% higher yield onbiomass than the control strain (49.4 and 40.0% TAG yields on biomass,respectively).

TABLE 4 Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) andyield (%) per used glucose after 35 hours cultivation in glucose mediumwith C/N ratio of 103 Yield TAG (% used Strain TAG (g/l) glucose) TAGmg/l/h Control 2.00 16.2 57.1 Y23/81-66 (ALD) 2.05 18.0 58.7 Y23/85-125(ALD + ACS) 2.11 19.3 60.3 Y23/86-92 (ACS) 2.17 17.4 61.9 Y23/95-98(PDAT) 2.22 19.4 63.5 Y23/81/95-18 (ALD + PDAT) 2.22 21.1 63.5

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations and ratesof production and triacylglycerol yield from used glucose or per dryweight with high C/N ratios.

Example 25. Aerobic Shake Flask Characterization of Strains M22/75-86(Ex. 15B), M22/96-1 (Ex. 17B), M22/94-24 (Ex. 16B), M22/75/98-9 (Ex. 19)and M22/94/98-19 (Ex. 20), in Glucose Medium with C/N Ratio of 40

Transformants were separately cultivated in 50 ml of mould C/N 40 medium(pH 5.5, 20 g glucose, 1.4 g yeast extract, 2.5 g KH₂PO₄, 0.3 g(NH₄)₂SO₄, 10 mg ZnSO₄*7H₂O, 2 mg CuSO₄*5H₂O, 10 mg MnSO₄, 0.5 gMgSO₄*7H₂O, 0.1 g CaCl₂, 20 mg FeCl₃*6H₂O per litre). Each flask (250ml) was inoculated with 1*10⁷ spores. The cultivations were maintainedat a temperature of 25° C. with shaking at 250 rpm. Samples for cell dryweight measurement, lipid extraction and HPLC analysis were withdrawnperiodically during cultivation. Mucor circinelloides wild type strainM22 was used as a control. Cell dry weight was determined by vacuumfiltration through disks of glass microfiber filters (Whatman, England)of 2 to 24 ml culture broth. After washing twice with distilled water,biomass was removed from the cloth using clean spatula and transferredto pre-dried, pre-weighed 2 ml microfuge tubes in which the mycelia weredried at 100° C. for 48 hours and weighed after cooling in a dessicator.HPLC analysis was carried out as described in Example 22. Lipidextraction and total lipid and triacylglycerol measurements as describedin Example 21.

After 46 hours cultivation (Table 5) transformants M22/75-86, M22/96-1,M22/94-24, M22/75/98-9 and M22/94/98-19 produced 21, 9, 18, 91 and 55%more triacylglycerol with higher rate than the control strain,respectively. The transformant M22/75-86, M22/96-1., M22/94-24,M22/75/98-9 and M22/94/98-19 had also 39 (20), 23 (10), 18 (13), 127(57) and 80 (25)% higher triacylglycerol yield on used glucose (onbiomass) than the control strain, respectively. Additionally thetransformants M22/75/98-9 and M22/94/98-19 had higher total lipidconcentration (0.93-1.09 g/l) and rate (20-24 mg/l/h) with yields onbiomass (26.1-31.4%) and on used glucose (6.29-7.52%) than the controlstrain (0.71 g/l, 15 mg/l/h, 24.6% and 4.09%, respectively).

TABLE 5 Triacylglycerol (TAG) and total lipid concentrations (g/l),rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) andused glucose after 46 hours cultivation in glucose medium with C/N ratioof 40 Yield Yield TAG TAG (% used TAG Strain TAG g/l (% CDW) glucose)mg/l/h Control 0.33 11.5 1.92 7.2 M22/75-86 (ALD) 0.40 13.8 2.67 8.8M22/96-1 (ACS) 0.36 12.6 2.36 7.9 M22/94-24 (ALD + ACS) 0.39 13.0 2.278.5 M22/75/98-9 (ALD + 0.63 18.1 4.35 13.7 PDAT) M22/94/98-19 0.51 14.43.46 11.1 (ALD + ACS + PDAT) Yield lipid Yield lipid (% used LipidStrain Lipid g/l (% CDW) glucose) mg/l/h Control 0.71 24.6 4.09 15M22/75-86 (ALD) 0.76 26.1 5.04 17 M22/96-1 (ACS) 0.70 24.2 4.53 15M22/94-24 (ALD + ACS) 0.71 23.5 4.10 15 M22/75/98-9 (ALD + 1.09 31.47.52 24 PDAT) M22/94/98-19 (ALD + 0.93 26.1 6.29 20 ACS + PDAT)

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol and total lipidconcentrations and rates of production and triacylglycerol and totallipid yields from used glucose or per dry weight.

Example 26. Aerobic Shake Flask Characterization of Strains M22/75-86(Ex. 15B), M22/96-1 (Ex. 17B), M22/94-24 (Ex. 16B), M22/75/98-9 (Ex. 19)and M22/94/98-19 (Ex. 20), in Glucose Medium with C/N Ratio of 66

Transformants were separately cultivated in 50 ml of mould C/N 66 medium(pH 5.5, 20 g glucose, 1.0 g yeast extract, 2.5 g KH₂PO₄, 0.1 g(NH₄)₂SO₄, 10 mg ZnSO₄*7H₂O, 2 mg CuSO₄*5H₂O, 10 mg MnSO₄, 0.5 gMgSO₄*7H₂O, 0.1 g CaCl₂, 20 mg FeCl₃*6H₂O per litre). Each flask (250ml) was inoculated with 1*10⁷ spores. The cultivations were maintainedat a temperature of 25° C. with shaking at 250 rpm. Samples for cell dryweight measurement, lipid extraction, enzyme activity measurement andHPLC analysis were withdrawn periodically during cultivation. Mucorcircinelloides wild type strain M22 was used as a control. Cell dryweight was determined as described in Example 25 and HPLC analysis wascarried out as described in Example 22. Lipid extraction and total lipidand triacylglycerol measurements as described in Example 21.

After 93 hours cultivation, when 2-6 g/l glucose was left, transformantsM22/75-86, M22/96-1, M22/94-24, M22/75+98-9 and M22/94+98-19 hadproduced 34, 30, 45, 186 and 214% more triacylglycerols with higher ratethan the control strain. The transformants M22/75-86, M22/96-1,M22/94-24, M22/75+98-9 and M22/94+98-19 also had 63 (24), 68 (30), 62(43), 229 (72) and 102 (104)% higher triacylglycerol yield on usedglucose (on biomass) than the control strain. Additionally, thetransformants produced more lipids (0.89-2.29 g/l) with higher rates(9.6-24.6 mg/l/h) and with higher yields on used glucose (6.90-17.68%)and on biomass (37.2-57.7%) than the control strain (0.71 g/l, 7.6mg/l/h, 4.82% and 30.2%, respectively).

TABLE 6 Triacylglycerol (TAG) and total lipid concentrations (g/l),rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) andused glucose after 93 hours cultivation in glucose medium with C/N ratioof 66. Yield Yield TAG TAG TAG (% used TAG Strain g/l (% CDW) glucose)mg/l/h Control 0.44 18.6 2.97 4.7 M22/75-86 (ALD) 0.59 23.0 4.84 6.4M22/96-1 (ACS) 0.57 24.1 4.99 6.1 M22/94-24 (ALD + ACS) 0.64 26.6 4.826.9 M22/75/98-9 1.26 31.9 9.76 13.6 (ALD + PDAT) M22/94/98-19 1.38 37.99.00 14.8 (ALD + ACS + PDAT) Yield lipid Yield lipid (% used LipidStrain Lipid g/l (% CDW) glucose) mg/l/h Control 0.71 30.2 4.8 7.6M22/75-86 (ALD) 0.96 37.2 7.8 10.3 M22/96-1 (ACS) 0.89 37.3 7.7 9.6M22/94-24 (ALD + ACS) 0.91 37.6 6.9 9.8 M22/75/98-9 (ALD + 2.29 57.717.7 24.6 PDAT) M22/94/98-19 2.00 55.1 13.1 21.5 (ALD + ACS + PDAT)

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol and total lipidconcentrations and rates of production and triacylglycerol and totallipid yields from used glucose or per dry weight with high C/N ratios.

Example 27. Microaerobic Shake Flask Characterization of StrainsY23/81-51 and Y23/81-66 (Ex. 3B), Y23/85-125 and Y23/85-128 (Ex. 7B),Y23/86-86 and Y23/86-92 (Ex. 5B), Y23/95-87 and Y23/95-109 (Ex. 6B) andY23/85/95-4 (Ex. 9), in Xylose Medium with C/N Ratio of 20

Transformants were separately cultivated in 50 ml of culture medium“xylose CN20” (pH 5.5, 20 g xylose, 0.3 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5 gNa₂HPO₄*2 H₂O, 1.5 g MgSO₄*7H₂O, 4.0 g Yeast extract, 51 mg CaCl₂, 8 mgFeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) wasinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 100 rpm. Samples for cell dry weight measurement,lipid extraction, enzyme activity measurement and HPLC analysis werewithdrawn periodically during cultivation. Cryptococcus curvatus wildtype strain Y23 was used as a control.

Lipid extraction and triacylglycerol concentration measurements werecarried out as described in Example 21. Cell dry weight was determinedand HPLC analysis was carried out as described in Example 22.

At the end of cultivation samples for acetaldehyde dehydrogenase andacetyl-CoA synthetase activity measurements were taken. Cells wereharvested by centrifugation and washed once with 100 mM Tris-HCl pH 7.0.Washed cells were stored at −20° C. Frozen cells were melted and washedonce with 100 mM Tris-HCl pH 7.0 and suspended in 100 mM Tris-HCl pH7.0, 1 mM DTT buffer containing EDTA-free protease inhibitors (Roche,USA). Cell disruption was carried out with 0.5 mm diameter glass beads(Sigma Chemicals Co, USA) in a Fast Prep homogenizer (Thermo Scientific,USA). Cell debris was removed by centrifugation and supernatant was usedin enzyme activity measurements. Acetaldehyde dehydrogenase andacetyl-CoA synthetase activity measurements were carried out with aKonelab Arena automatic analyzer (Thermo Scientific, Finland). Theacetaldehyde dehydrogenase reaction mixture contained (finalconcentration) 50 mM potassium phosphate pH 7.0, 15 mM pyrazole, 0.4 mMDTT, 10 mM MgCl₂, 0.4 mM NADP and cell extract. The reaction was startedwith 0.1 mM acetaldehyde. The formation of NADPH was followed at 340 nm.One unit was defined as the amount of formation of 1 μmol of NADPH permin. The acetyl-CoA synthetase reaction mixture contained (finalconcentration) 100 mM Tris-HCl pH 7.5, 10 mM L-malate pH 7.5, 0.2 mMCoenzyme A, 8 mM ATP, 1 mM NAD, 10 mM MgCl₂, 3 U/ml malatedehydrogenase, 0.4 U/ml citrate synthase and cell extract. The reactionwas started with 100 mM potassium acetate. The formation of NADH wasfollowed at 340 nm. One unit was defined as the amount of formation of 1μmol of NADH per min.

After 24 hours cultivation the transformants Y23/81, Y23/85, Y23/86,Y23/95 and Y23/85/95 produced 13-31% more triacylglycerol with 5-38%higher yield on biomass than the control strain. The transformantsY23/81, Y23/86, Y23/95 and Y23/85/95 also had 4-12% higher yields onused xylose than the control strain. The transformants having ALD (ACS)encoding gene expressed had 21.5 to 42.7 (1.5 to 1.9) times higher ALD(ACS) activity than the control strain.

TABLE 7 Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) andyields (%) per biomass (CDW; cell dry weight) and used xylose after 24hours microaerobic cultivation in xylose medium with C/N ratio of 20 TAGYield TAG Yield TAG (% TAG Strain (g/l) (% CDW) used xylose) mg/l/hControl 0.16 4.33 3.46 6.8 Y23/81 (ALD) 0.20 5.98 3.61 8.2 Y23/85 (ALD +ACS) 0.18 4.54 3.46 7.3 Y23/86 (ACS) 0.21 5.33 3.86 8.9 Y23/95 (PDAT)0.20 5.55 3.68 8.4 Y23/85/95 0.20 5.55 3.72 8.2 (ALD + ACS + PDAT)

TABLE 8 Relative acetaldehyde dehydrogenase (ALD) and acetyl-CoAsynthetase (ACS) activities in microaerobic cultivation in xylose withC/N ratio of 20 compared to the control strain ALD and ACS activitiesStrain ALD ACS Control 1 1 Y23/81 (ALD) 42.7 1.2 Y23/85 (ALD + ACS) 27.31.9 Y23/86 (ACS) 4.3 1.5 Y23/95 (PDAT) 2.3 1.2 Y23/85/95 (ALD + ACS +PDAT) 21.5 1.5

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations and ratesof production and triacylglycerol yields from used xylose or per dryweight with low C/N ratios. Additionally, the example shows thatacetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS) enzymesare expressed in active forms.

Example 28. Aerobic Shake Flask Characterization of Strains Y23/81-8 and59 (Ex. 3B), Y23/85-119 and 128 (Ex. 7B), Y23/86-86 and 92 (Ex. 5B),Y23/95-99 and 109 (Ex. 6B), Y23/81/95-18 and 42 (Ex. 8) and Y23/85/95-4and 68 (Ex. 9), in Xylose Medium with C/N Ratio of 103

Transformants were separately cultivated in 50 ml of Yeast xyloseculture medium IV (pH 5.5, 20 g xylose, 0.15 g (NH₄)₂SO₄, 7.0 g KH₂PO₄,2.5 g Na₂HPO₄*2H₂O, 1.5 g MgSO₄*7H₂O, 0.45 g Yeast extract, 51 mg CaCl₂,8 mg FeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml)was inoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 250 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis were withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 was used as acontrol. Cell dry weight was determined and HPLC analysis was carriedout as described in Example 22. Lipid extraction and triacylglycerolmeasurements as described in Example 21.

After 52 hours cultivation the transformants Y23/81, Y23/85, Y23/86,Y23/95, Y23/81/95 and Y23/85/95 had produced 23, 19, 50, 52, 40 and 75%more triacylglycerol than the control strain. The transformants Y23/81,Y23/85, Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had also 35, 47, 59, 60,103 and 111% higher triacylglycerol yield on biomass and 11, 11, 15, 28,66 and 63% higher triacylglycerol yield on used xylose than the controlstrain. Additionally the transformants Y23/81, Y23/85, Y23/86, Y23/95,Y23/81/95 and Y23/85/95 had higher lipid concentration (2.40-2.90 g/l)and rate (46-56 mg/l/h) with higher yield on biomass (56.5-78.8%) thanthe control strain (2.35 g/l, 45 mg/l/h and 52.2%, respectively). Thetransformants Y23/81/95 and Y23/85/95 had also higher lipid yield onused xylose (29.0-31.6%) than the control strain (25.6%).

TABLE 9 Triacylglycerol (TAG) and total lipid concentrations (g/l),rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) after52 hours cultivation in xylose medium with C/N ratio of 103.Triacylglycerol yield (%) from used xylose is also indicated. Yield TAGYield TAG (% used TAG Strain TAG (g/l) (% CDW) xylose) mg/l/h Control0.60 13.3 6.52 19.0 Y23/81 (ALD) 0.83 17.9 7.23 26.4 Y23/85 (ALD + ACS)0.79 19.6 7.26 25.0 Y23/86 (ACS) 0.90 21.1 7.52 28.5 Y23/95 (PDAT) 0.9121.3 8.37 28.8 Y23/81/95 0.84 27.0 10.8 26.6 (ALD + PDAT) Y23/85/95 1.0528.0 10.7 33.3 (ALD + ACS + PDAT) Yield lipid Strain Lipid (g/l) (% CDW)Lipid mg/l/h Control 2.35 52.2 45 Y23/81 (ALD) 2.90 62.3 56 Y23/85(ALD + ACS) 2.53 62.9 49 Y23/86 (ACS) 2.58 60.6 50 Y23/95 (PDAT) 2.4056.5 46 Y23/81/95 (ALD + PDAT) 2.45 78.8 47 Y23/85/95 (ALD + ACS + PDAT)2.85 76.0 55

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol and total lipidconcentrations and rates of production and triacylglycerol and totallipid yields per dry weight and triacylglycerol yields from used xylosewith high C/N ratios.

Example 29. Aerobic Shake Flask Characterization of Strains M22/75-80(Ex. 15B), M22/96-6 (Ex. 17B), M22/98-16 (Ex. 18B), M22/94-16 (Ex. 16B),M22/75/98-7 (Ex. 19) and M22/94/98-22 (Ex. 20), in Xylose Medium withC/N Ratio of 66

Transformants were separately cultivated in 50 ml of mould xylose C/N 66medium (pH 5.5, 20 g xylose, 1.0 g yeast extract, 2.5 g KH₂PO₄, 0.1 g(NH₄)₂SO₄, 10 mg ZnSO₄*7H₂O, 2 mg CuSO₄*5H₂O, 10 mg MnSO4, 0.5 gMgSO₄*7H₂O, 0.1 g CaCl₂, 20 mg FeCl₃*6H₂O per litre). Each flask (250ml) was inoculated with 1*10⁷ spores. The cultivations were maintainedat a temperature of 25° C. with shaking at 250 rpm. Samples for cell dryweight measurement, lipid extraction, enzyme activity measurement andHPLC analysis were withdrawn periodically during cultivation. Mucorcircinelloides wild type strain M22 was used as a control. Cell dryweight was determined as described in Example 25, and HPLC analysis wascarried out as described in Example 22. Lipid extraction and total lipidand triacylglycerol measurements as described in Example 21.

After 143 hours cultivation the transformants M22/75-80, M22/96-6,M22/98-16, M22/75/98-7 and M22/94/98-22 had higher TAG concentration(0.33-0.48 g/l TAG) compared to the control strain (0.28 g/l TAG). Theall transformants had also higher TAG yield (%) per biomass(13.71-17.78%) than the control strain (12.15%) and the transformantsM22/96-6, M22/98-16, M22/75/98-7 and M22/94/98-22 had also higher TAGyield (%) per used xylose (4.83-6.23%) than the control strain (3.97%).Additionally the transformants M22/75/98-7 and M22/94/98-22 had higherlipid concentration (0.79-1.13 g/l), rate (9.2-10.8 mg/l/h) and lipidyields on biomass (48.9-55.6%) and on used xylose (17.1-18.3%) than thecontrol strain (0.92 g/l, 6.4 mg/l/h, 40.0% and 13.0%, respectively).

TABLE 10 Triacylglycerol (TAG) and total lipid concentrations (g/l),rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) andused xylose after 143 hours cultivation in xylose medium with C/N ratioof 66 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) xylose)mg/l/h Control 0.28 12.2 3.97 1.9 M22/75-80 (ALD) 0.33 14.8 3.45 2.3M22/96-6 (ACS) 0.33 15.3 5.40 2.3 M22/94-16 (ALD + ACS) 0.27 13.7 3.671.9 M22/98-16 (PDAT) 0.48 13.7 4.83 3.3 M22/75/98-7 0.48 17.8 6.23 3.3(ALD + PDAT) M22/94/98-22 0.45 16.1 5.31 3.1 (ALD + ACS + PDAT) Yieldlipid Lipid Yield lipid (% used Lipid Strain (g/l) (% CDW) xylose)mg/l/h Control 0.92 40.0 13.0 6.4 M22/75-80 (ALD) 1.00 44.8 10.5 7.0M22/96-6 (ACS) 0.90 41.5 14.7 6.3 M22/94-16 (ALD + ACS) 0.82 41.6 11.15.7 M22/98-16 (PDAT) 1.62 46.4 16.4 11.3 M22/75/98-7 (ALD + PDAT) 1.3248.9 17.1 9.2 M22/94/98-22 (ALD + 1.54 55.6 18.3 10.8 ACS + PDAT)

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol and total lipidconcentrations and rates of production and triacylglycerol and totallipid yields from used xylose or per dry weight with high C/N ratios.

Example 30. Production of Triacylglycerol or Lipid by Strains of C.curvatus Modified by Addition of Genes Encoding ALD and PDAT(Y23/81/95-18, Ex. 8) or ALD and PDAT and ACS (Y23/85/95-4 Ex. 9) inHigh Cell Density Cultures Grown on Glucose with C/N Ratio of 80

Transformants (Y23/85/95-4 and Y23/81/95-18) were separately cultivatedin Multifors bioreactors (max. working volume 500 ml, Infors HT,Switzerland) at pH 4.0, 30° C., in 500 ml medium containing 90 to 96 gglucose, 2.56 g (NH₄)₂SO₄, 1.2 g KH₂PO₄, 0.3 g Na₂HPO₄.2H₂O, 1.5 gMgSO₄.7H₂O, 0.1 g CaCl₂.6H₂O, 5.26 mg citric acid.H₂O, 5.26 mgZnSO₄.7H₂O, 0.1 mg MnSO₄.4H₂O, 0.5 mg CoCl₂.6H₂O, 0.26 mg CuSO₄.5H₂O,0.1 mg Na₂MoO₄.2H₂O, 1.4 mg FeSO₄.7H₂O, 0.1 mg H₃BO₄, 0.05 mg D-biotin,1.0 mg CaPantothenate, 5.0 mg nicotinic acid, 25 mg myoinositol, 1.0 mgthiamine.HCl, 1.0 mg pyridoxine.HCl and 0.2 mg p-aminobenzoic acid perlitre. The pH was maintained constant by addition of 1 M KOH or 1 MH₂PO₄. Cultures were agitated at 1000 rpm (2 Rushton turbine impellors)and aerated at 2 volumes air per volume culture per minute (vvm). ClerolFBA 3107 antifoaming agent (Cognis, SaintFargeau-Ponthierry CedexFrance, 1 ml 1⁻¹) was added to prevent foam accumulation. Bioreactorswere inoculated to initial OD₆₀₀ of 0.5 to 4.0 with cells grown in thesame medium (substituting 1.5 g urea per litre for (NH₄)₂SO₄ andomitting the CaCl₂.6H₂O) in 50 ml volumes in 250 ml flasks at 30° C.with shaking at 200 rpm for 24 to 42 h. Samples for cell dry weightmeasurement, lipid extraction and HPLC analysis were withdrawnperiodically during cultivation. Cryptococcus curvatus wild type strainY23 was used as the control. For measurement of the yield oftriacylglycerol on glucose or biomass, some control cultures contained58 to 134 g glucose 1⁻¹.

Lipid extraction and triacylglycerol concentration measurements werecarried out as described in Example 21. Cell dry weight was determinedby centrifuging 0.5 to 2.0 ml culture broth in pre-dried, pre-weighed 2ml microfuge tubes. After washing twice with 1.8 ml distilled water, thecell pellet was dried at 100° C. for 48 h and weighed after cooling in adessicator. HPLC analyses were carried out as described in Example 22.

Table 11 shows that a transformant containing the genes for ALD and PDATproduced 23% more triacylglycerol than Y23, with a 24% increase in theyield on glucose consumed when cells were cultivated to high celldensity in bioreactor cultures. A transformant containing the genes forALD, ACS and PDAT produced 15% more triacylglycerol than Y23, with a 12%increase in the yield on glucose consumed.

TABLE 11 Triacylglycerol produced in pH controlled bioreactor culture ofY23 and transformants of Y23 expressing ALD + PDAT or ALD + ACS + PDAT,with glucose as carbon source and C/N 80. Data is the average of 2(transformants) or 4 to 9 (Y23) cultures ± standard error of the mean.Percentage increase is shown in parenthesis Yield TAG Strain TAG (g/l)(% glucose consumed) Y23 17.3 ± 0.3 18.8 ± 1.0 Y23/81/95-18 (ALD + PDAT)21.3 ± 0.8 23.3 ± 1.0 (23%) (24%) Y23/85/95-4 19.9 ± 0.1 21.1 ± 0.1(ALD + ACS + PDAT) (15%) (12%)

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations andtriacylglycerol yields from used glucose in high cell density cultures.

Example 31. Production of Triacylglycerol or Lipid by Strains of C.curvatus Modified by Addition of Genes Encoding ALD and PDAT(Y23/81/95-18, Ex. 8) or ALD and PDAT and ACS (Y23/85/95-4, Ex. 9) inHigh Cell Density Cultures Grown on Xylose with C/N Ratio of 80

Transformants (Y23/85/95-4 and Y23/81/95-18) were separately cultivatedin Multifors bioreactors as described in Example 30. The mediumcontained 92 to 118 g xylose per litre, instead of glucose. Bioreactorswere inoculated to initial OD₆₀₀ of 17 to 24 with cells grown in lownitrogen medium with glucose as carbon source in the Multiforsbioreactors at 30° C., as described for lipid production in Example 30.Alternatively, some cultures of Y23 were inoculated with cells grown inflasks, as described in Example 30, to initial OD₆₀₀ 0.2 to 0.5. Samplesfor cell dry weight measurement, lipid extraction and HPLC analysis werewithdrawn periodically during cultivation. Cryptococcus curvatus wildtype strain Y23 was used as the control.

Lipid extraction and total lipid and triacylglycerol concentrationmeasurements were carried out as described in Example 21. Cell dryweight was determined as described in Example 30. HPLC analyses werecarried out as described in Example 22.

Table 12 shows that a transformant containing the genes for ALD and PDATproduced 7% more triacylglycerol than Y23, with a 17% increase in theyield on xylose consumed and a 3% increase in the yield on biomass whencells were cultivated to high cell density in bioreactor cultures. Atransformant containing the genes for ALD, ACS and PDAT produced only 1%more triacylglycerol than Y23, but showed 13% increase in the yield onxylose consumed and 4% increase in yield on biomass.

TABLE 12 Triacylglycerol produced in pH controlled bioreactor culture ofY23 and transformants of Y23 expressing ALD + PDAT or ALD + ACS + PDAT,with lose as carbon source and C/N 80. Data is the average of 2(transformants) or 4 (Y23) cultures, ± standard error of the mean.Percentage increase is shown in parenthesis. Yield TAG Yield TAG (%Strain TAG (g/l) (% CDW) xylose consumed) Y23 17.9 ± 1.4 46.9 ± 4.0 17.2± 1.2 Y23/81/95-18 19.1 ± 0.3 48.4 ± 5.7 20.2 ± 0.5 (ALD + PDAT) (7%)(3%) (17%) Y23/85/95-4 18.1 ± 0.3 49.0 ± 3.4 19.5 ± 0.0 (ALD + ACS +PDAT) (1%) (4%) (13%)

Table 13 shows that a transformant containing the genes for ALD and PDATproduced 10% more lipid than Y23, with a 21% increase in the yield onxylose consumed and a 2% increase in the yield on biomass when cellswere cultivated to high cell density in bioreactor cultures. Atransformant containing the genes for ALD, ACS and PDAT produced 3% morelipid than Y23, with 16% higher yield on xylose consumed and a 3%increase in yield on biomass.

TABLE 13 Lipid produced in pH controlled bioreactor culture of Y23 andtransformants of Y23 expressing ALD + PDAT or ALD + ACS + PDAT, withxylose as carbon source and C/N 80. Data is the average of 2(transformants) or 4 (Y23) cultures, ± standard error of the mean.Percentage increase is shown in parenthesis. Yield lipid Yield lipid (%Strain Lipid (g/l) (% CDW) xylose consumed) Y23 20.5 ± 1.3 55.9 ± 7.219.8 ± 2.5 Y23/81/95-18 (ALD + PDAT) 22.5 ± 0.6 57.0 ± 1.9 23.8 ± 0.8(10%) (2%) (21%) Y23/85/95-4 21.2 ± 0.1 57.5 ± 1.0 22.9 ± 0.3 (ALD +ACS + PDAT)  (3%) (3%) (16%)

This example shows that expression of ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol and total lipidconcentrations and triacylglycerol and total lipid yields from usedxylose or per dry weight in high cell density cultures.

Example 32. Production of Triacylglycerol or Lipid by Strain of M.circinelloides Modified by Addition of Genes Encoding ALD and PDAT andACS (M22/94/98-19, Ex. 20) in pH Controlled Bioreactor Cultures Grown onGlucose with C/N Ratio of 60

Transformant (M22/94/98-19) was cultivated in Braun Biostat® CTbioreactors (2.5 max working volume, B. Braun Biotech International,Sartorius AG, Germany) at pH 5.0, 30° C., in 1.0 to 1.2 l mediumcontaining 53 g glucose, 1.57 g (NH₄)₂SO₄, 2.5 g KH₂PO₄, 0.2 gMgSO₄.7H₂O, 0.1 g CaCl₂.6H₂O, 5.26 mg citric acid.H₂O, 5.26 mgZnSO₄.7H₂O, 0.1 mg MnSO₄.4H₂O, 0.5 mg CoCl₂.6H₂O, 0.26 mg CuSO₄.5H₂O,0.1 mg Na₂MoO₄.2H₂O, 1.4 mg FeSO₄.7H₂O, 0.1 mg H₃BO₄, 0.005 mg D-biotin,and 0.05 mg thiamine.HCl per litre. The pH was maintained constant byaddition of 1 M KOH or 1 M H₂PO₄. Cultures were agitated at 600 rpm (2Rushton turbine impellors) and aerated at 1 volume air per volumeculture per minute (vvm). Polypropylene glycol (mixed molecular weightscontaining Fluka P1200, Fluka P2000 and Henkel Performance ChemicalsFoamaster in a ratio of 4:4; 1, 1 ml 1⁻¹) was added to prevent foamaccumulation. Bioreactors were inoculated to an initial biomassconcentration of approximately 100 mg 1⁻¹ with mycelia grown in the samemedium with the following modifications: 15 g glucose 1⁻¹, enough(NH₄)₂SO₄ to provide a C/N ratio of 16.2, and additionally 4.0 g agar1⁻¹. Pre-cultures were grown in 50 ml volumes in 250 ml flasks at 30° C.with shaking at 200 rpm for 42 to 72 h. Samples for cell dry weightmeasurement, lipid extraction and HPLC analysis were withdrawnperiodically during cultivation. Biomass was separated from the culturesupernatant by filtration under vacuum. Mucor circinelloides wild typestrain M22 was used as the control.

Lipid extraction and total lipid and triacylglycerol concentrationmeasurements were carried out as described in Example 21. Cell dryweight was determined as described in Example 25, except that disposablecleaning cloth (X-tra, 100% viscose household cleaning cloth, InexPartners Oy, Helsinki) was used in vacuum filtration. HPLC analyses werecarried out as described in Example 22.

Table 14 shows that a transformant containing the genes for ALD, ACS andPDAT produced 17% more triacylglycerol than Y23, with 8% increase in theyield on glucose consumed, when cells were cultivated in bioreactorcultures.

TABLE 14 Triacylglycerol produced in pH controlled bioreactor culturesof M22 and transformant of M22 expressing ALD + ACS + PDAT, with glucoseas carbon source and C/N 60. Percentage increase is shown inparenthesis. Yield TAG (% Strain TAG (g/l) glucose consumed) M22 9.821.9 M22/94/98-19 11.5 (17%) 23.6 (8%) (ALD + ACS + PDAT)

Table 15 shows that a transformant containing the genes for ALD, ACS andPDAT produced 44% more lipid than M22, with 55% higher yield on glucoseconsumed and 26% increase in yield of lipid on biomass, when cells werecultivated in bioreactor cultures.

TABLE 15 Lipid produced in pH controlled bioreactor cultures of M22 andtransformant of M22 expressing ALD + ACS + PDAT, with glucose as carbonsource and C/N 60. Percentage increase is shown in parenthesis. Yieldlipid Yield lipid (% Strain Lipid (g/l) (% CDW) glucose consumed) M2210.4 59.4 ± 2.8 19.7 M22/94/98-19 14.9 (44%) 75.4 ± 3.5 30.5 (55%)(ALD + ACS + PDAT) (26%)

This example shows that expression of ALD6, ACS2 and PDAT genes enhancedtriacylglycerol and total lipid concentrations and triacylglycerol andtotal lipid yields from used glucose or total lipid yield per dry weightin high cell density cultures.

Example 33. Production of Triacylglycerol or Lipid by Strain of M.circinelloides Modified by Addition of Genes Encoding ALD and PDAT andACS (M22/94/98-19, Ex. 20) in pH Controlled Bioreactor Cultures Grown onXylose with C/N Ratio of 60

Transformant (M22/94/98-19) was cultivated in Braun Biostat CTbioreactors as described in Example 32. The medium contained 44 to 56 gxylose per litre, instead of glucose cultures were supplemented with 1 gpeptone per litre and the (NH₄)₂SO₄ concentration was reduced to 1.12 gper litre. Samples for cell dry weight measurement, lipid extraction andHPLC analysis were withdrawn periodically during cultivation. Mucorcircinelloides wild type strain M22 was used as the control.

Lipid extraction and total lipid and triacylglycerol concentrationmeasurements were carried out as described in Example 21. Cell dryweight was determined as described in Example 32. HPLC analyses werecarried out as described in Example 22.

Table 16 shows that a transformant containing genes for ALD, ACS andPDAT produced 11% more triacylglycerol than M22, with 10% increasedyield on xylose consumed and 9% increased yield on biomass in pHcontrolled bioreactor cultures.

TABLE 16 Triacylglycerol produced in pH controlled bioreactor culturesof M22 and transformant of M22 expressing ALD + ACS + PDAT, with xyloseas carbon source and C/N 60. Percentage increase is shown inparenthesis. Yield TAG TAG Yield TAG (% xylose Strain (g/l) (% CDW)consumed) M22 5.6 46.5 ± 2.7 22.9 M22/94/98-19 6.2 50.9 ± 1.4 25.1(ALD + ACS + PDAT) (11%) (9%) (10%)

Table 17 shows that a transformant containing the genes for ALD and ACSand PDAT produced 24% more lipid than M22 and the yield of lipid onbiomass was 22% higher than in M22, when mycelia were grown on xylose.The yield of lipid on xylose consumed was increased 18%.

TABLE 17 Lipid produced in pH controlled bioreactor culture of M22 andtransformant of M22 expressing ALD + ACS + PDAT, with xylose as carbonsource and C/N 60. Percentage increase is shown in parenthesis. Yieldlipid Lipid Yield lipid (% xylose Strain (g/l) (% CDW) consumed) M22 5.849.2 ± 4.6 24.1 M22/94/98-19 7.1 58.2 ± 0.6 28.7 (ALD + ACS + PDAT)(22%) (18%) (19%)

This example shows that expression of ALD6, ACS2 and PDAT genes enhancedtriacylglycerol and total lipid concentrations and triacylglycerol andtotal lipid yields from used xylose or per dry weight in high celldensity cultures.

Example 34. Construction of a Plasmid Containing a Marker Gene Under theControl of an Endogenous Promoter and Terminator and a PyruvateDecarboxylase (PDC) Encoding Gene Under the Control of an EndogenousPromoter and Terminator

A pyruvate decarboxylase (PDC) encoding gene, such as PDC1 from S.cerevisiae (SEQ ID NO:94) which encodes the amino acid sequence of SEQID NO:95 is codon optimised. The codon optimised PDC encoding gene withflanking SbfI restriction sites is digested with SbfI and ligated to aplasmid containing an endogenous promoter and terminator, such asplasmid pKK77pre. The resulting plasmid which contains the PDC encodinggene under the control of the endogenous promoter and terminator will belinearised e.g. with BamHI and ligated with a fragment containing themarker gene under the control of the endogenous promoter and terminator.Such fragment can be obtained e.g. by digesting a plasmid pKK67 withBamHI and XmnI. The resulting plasmid contains the PDC encoding geneunder the control of the endogenous promoter and terminator and themarker gene under the control of the endogenous promoter and terminator.

Example 35. Generation of Genetically Modified Strain with an IntegratedPDC Together with ALD6 and/or ACS2 Encoding Genes and Marker Genes byTransforming Genetically Modified Strains with Plasmid Containing PDCEncoding Gene (Ex 34)

The plasmid containing the PDC encoding gene (Ex. 34) is restricted e.g.with NotI and PspOMI, and the resulting linear DNA containing the PDCencoding gene under the control of the endogenous promoter andterminator and the marker gene under the control of the endogenouspromoter and terminator is used to transform e.g. a genetically modifiedstrain Y23/81-51 (Ex. 3B), Y23/86-92 (Ex. 5B) or Y23/85-128 (Ex. 7B) byelectroporation or a genetically modified strain M22/75-86 (Ex. 15B),M22/96-1 (Ex. 17B) or M22/94-31 (Ex 16B) using the transformation methoddescribed in Example 15B. The transformed cells are screened e.g. forantibiotic resistance. Several transformed colonies are analysed at DNAlevel by PCR.

Example 36. Aerobic Shake Flask Characterization of Strains HarbouringPDC Together with ALD6 and/or ACS2 Encoding Genes and Marker Genes inGlucose or Xylose Medium with Different C/N Ratios

Transformants are separately cultivated in 50 ml of culture medium suchas described in Examples 22, 23, 24, 27 or 28. Each flask (250 ml) isinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations are maintained at a temperature of 30°C. with shaking at 250 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis are withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 is used as acontrol. Cell dry weight is determined and HPLC analysis is carried outas described in Example 22. Lipid extraction and triacylglycerolmeasurements as described in Example 21. Alternatively transformants areseparately cultivated in 50 ml of culture medium such as described inExamples 25, 26 or 29. Each flask (250 ml) is inoculated with 1*10⁷spores. The cultivations are maintained at a temperature of 25° C. withshaking at 250 rpm. Samples for cell dry weight measurement, lipidextraction and HPLC analysis are withdrawn periodically duringcultivation. Mucor circinelloides wild type strain M22 is used as acontrol. Cell dry weight is determined as described in Example 25 andHPLC analysis is carried out as described in Example 22. Lipidextraction and triacylglycerol measurements as described in Example 21.

The transformants harbouring PDC together with ALD6 and/or ACS2 encodinggenes produce more triacylglycerol than the control strain. Additionallythe transformants harbouring PDC together with ALD6 and/or ACS2 encodinggene have higher triacylglycerol yield on used carbon than the controlstrain.

Example 37A. Construction of a Plasmid (pKK101) Containing the G418Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1Terminator and the S. cerevisiae PDC1 Encoding Gene Under the Control ofthe CcTPI1 Promoter and the CcTEF1 Terminator

The plasmid Y4_TPIp-PDC-TEFt (Geneart AG, Germany) contains a S.cerevisiae PDC1 (SEQ ID NO:95) encoding gene which has been codonoptimized according to Ustilago maydis yeast codon usage (SEQ ID NO:96)with the CcTPI1 promoter and the CcTEF1 terminator. PlasmidY4_TPIp-PDC-TEFt was digested with EcoRI. A 2893 bp fragment was gelisolated and ligated to a 5381 bp fragment obtained by digesting aplasmid designated pKK67 (Ex. 2B) with EcoRI. The resulting plasmid wasdesignated as pKK101 (FIG. 12). The plasmid pKK101 contains a S.cerevisiae PDC1 encoding gene under the control of the CcTPI1 promoterand the CcTEF1 terminator and the E. coli G418 resistance gene under thecontrol of the CcTEF1 promoter and the CcTPI1 terminator.

Example 37B. Generation of Genetically Modified Strains with anIntegrated PDC Encoding Gene and G418 Resistance Gene by TransformingWild-Type C. curvatus and Genetically Modified Strains Y23/81-66 (Ex.3B), Y23/85-125 (Ex. 7B), Y23/86-86 (Ex. 5B), Y23/95-99 (Ex. 6B),Y23/81/95-18 (Ex. 8) and Y23/85/95-4 (Ex. 9) with Digested PlasmidpKK101 (FIG. 12, Ex. 37A)

Plasmid pKK101 was restricted with SacII and PspOMI, and the resultinglinear DNA was used to transform wild-type C. curvatus strain ATCC20509designated as Y23 and genetically modified strains Y23/81-66,Y23/85-125, Y23/86-86, Y23/95-99, Y23/81/95-18 and Y23/85/95-4 byelectroporation. The transformed cells were screened for G418resistance. Several G418 resistance colonies were analysed at DNA levelby PCR. The transformants originating from the transformation ofwild-type C. curvatus strain ATCC20509 designated as Y23 and geneticallymodified strains Y23/81-66, Y23/85-125, Y23/86-86, Y23/95-99,Y23/81/95-18 and Y23/85/95-4 with SacII and PspOMI cut pKK101 andcontaining the S. cerevisiae PDC1 encoding gene under the control of theCcTPI1 promoter and the CcTEF1 terminator were designated as Y23/101-55,Y23/101-57, Y23/101-59, Y23/81/101-4, Y23/85/101-13, Y23/85/101-14,Y23/85/101-19, Y23/86/101-23, Y23/95/101-1, Y23/95/101-2,Y23/81/95/101-20, Y23/85/95/101-7 and Y23/85/95/101-8.

Example 38A. Construction of a Plasmid (pKK102) Containing the CeruleninResistance Gene Under the Control of the McPGK1 Promoter and the McTPI1Terminator and the S. cerevisiae PDC1 Encoding Gene Under the Control ofthe McPGK1 Promoter and the McTEF1 Terminator

The plasmid M22_PGKp-PDC-TEFt (Geneart AG, Germany) contains a S.cerevisiae PDC1 (SEQ ID NO:95) encoding gene which has been codonoptimized according to Rhizopus oryzae filamentous fungus codon usage(SEQ ID NO:97) with the McPGK1 promoter and the McTEF1 terminator.Plasmid M22_PGKp-PDC-TEFt was digested with SalI. A 3363 bp fragment wasgel isolated and ligated to a 6239 bp fragment obtained by digesting aplasmid designated pKK92 (Ex. 14B) with SalI. The resulting plasmid wasdesignated as pKK102 (FIG. 13). The plasmid pKK102 contains a S.cerevisiae PDC1 encoding gene under the control of the McPGK1 promoterand the McTEF1 terminator and the g cerevisiae cerulenin resistance geneunder the control of the McPGK1 promoter and the McTPI1 terminator.

Example 38B. Generation of a Genetically Modified Mucor Circinelloides(M22/94/102) with Integrated ALD6, ACS1 and PDC1 Encoding Genes andHygromycin and Cerulenin Resistance Genes by Transforming GeneticallyModified Strain M22/94-12 (Ex. 16B) with Digested Plasmid pKK102 (FIG.13, Ex. 38A)

Plasmid pKK102 was restricted with SacII and PspOMI, and the resultinglinear DNA was used to transform the genetically modified strainM22/94-12, using the transformation method described in Example 15B. Thetransformed cells were screened for cerulenin resistance. Severalcerulenin resistant colonies were analysed at DNA level by PCR. Thetransformant originating from the transformation of the geneticallymodified strain M22/94-12 with SacII and PspOMI cut pKK102 andcontaining the S. cerevisiae PDC1 encoding gene under the control of theMcPGK1 promoter and the McTEF1 terminator was designated asM22/94/102-31.

Example 39. Aerobic Shake Flask Characterization of Strains Y23/101-59(Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-14 (Ex. 37B),Y23/86/101-23 (Ex. 37B), Y23/95/101-2 (Ex. 37B), Y23/81/95/101-20 (Ex.37B) and Y23/85/95/101-9 (Ex. 37B) in Glucose Medium with C/N Ratio of153

Transformants were separately cultivated in 50 ml of Glucose-CN153medium (pH 5.5, 30 g glucose, 0.15 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5 gNa₂HPO₄*2 H₂O, 1.5 g MgSO₄*7H₂O, 0.45 g Yeast extract, 51 mg CaCl₂, 8 mgFeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) wasinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 250 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis were withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 was used as acontrol. Cell dry weight was determined and HPLC analysis was carriedout as described in Example 22. Lipid extraction and triacylglycerolmeasurements as described in Example 21. Lipid extractions from theculture medium i.e. from the supernatant samples recovered aftercentrifugation of the cell lipid extraction samples or dry weightmeasurement samples were carried out as follows. To 150 μl ofsupernatant sample 150 μl of 0.9% NaCl and 150-1500 μl ofchloroform:methanol (2:1) was added. The sample was vortexed 2 minutesand sample was incubated at room temperature at 30 min. After incubationsample was centrifuged 10 000 rpm for 3 min at RT. The lower phase wasrecovered into microfuge tubes, dried and redissolved in 1.5 ml ofchloroform:methanol (2:1) and stored at −20° C. prior triacylglycerolmeasurements as described in Example 21. Alternatively, dried lipidpellet was redissolved directly in isopropanol and triacylglycerol wasmeasured as described in Example 21.

After 47 hours cultivation (Table 18), when 17 to 18 g/l glucose wasleft, Y23/101-59, Y23/85/101-14, Y23/86/101-23, Y23/95/101-2,Y23/81/95/101-20 and Y23/85/95/101-8 transformants produced 52, 78, 9,27, 32 and 48% more triacylglycerol with higher rate, respectively, thanthe control strain in glucose medium. Also triacylglycerol yields onbiomass and per used glucose were 33 to 83% and 35 to 85% higher intransformants Y23/101-59, Y23/85/101-14, Y23/86/101-23, Y23/95/101-2,Y23/81/95/101-20 and Y23/85/95/101-8 than the control strain,respectively.

TABLE 18 Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) andyield (%) per biomass (CDW) and used glucose in the yeast cells after 47hours cultivation in glucose medium with C/N ratio of 153 Yield TAG TAGYield TAG (% used TAG Strain (g/l) (% CDW) glucose) mg/l/h Control 1.3022.5 10.9 28 Y23/101-59 (PDC) 1.98 37.1 16.3 42 Y23/85/101-14 2.31 41.120.2 49 (PDC + ALD + ACS) Y23/86/101-23 1.42 29.8 15.4 30 (PDC + ACS)Y23/95/101-2 1.65 30.9 14.7 35 (PDC + PDAT) Y23/81/95/101-20 1.72 36.416.4 37 (PDC + ALD + PDAT) Y23/85/95/101-8 1.93 36.6 18.1 41 (PDC +ALD + ACS + PDAT)

After 94 hours cultivation (Table 19A), when 6 to 11 g/l glucose wasleft, Y23/101-59, Y23/85/101-14, Y23/81/95/101-20 and Y23/85/95/101-8transformants produced 5, 27, 8 and 12% more triacylglycerol with higherrate, respectively, than the control strain in glucose medium. Alsotriacylglycerol yields on biomass and per used glucose were 9 to 33% and7 to 38% higher in transformants Y23/101-59, Y23/81/101-4,Y23/85/101-14, Y23/86/101-23, Y23/95/101-2, Y23/81/95/101-20 andY23/85/95/101-8 than the control strain, respectively. Additionally,triacylglycerol concentration in the culture medium in the cultivationswith the transformants Y23/81/101-4 and Y23/86/101-23 was 525 and 350%higher, respectively, than in the cultivation with the control strain(Table 19B). Additionally, the total triacylglycerol yields per usedglucose calculated from the intracellular triacylglycerol concentrationand the triacylglycerol concentration detected from the culture mediumwere 13 to 38% higher with the transformants than with the controlstrain.

TABLE 19A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) andyield (%) per biomass (CDW) and used glucose in the yeast cells after 94hours cultivation in glucose medium with C/N ratio of 153 Yield TAG TAGYield TAG (% used TAG Strain (g/l) (% CDW) glucose) mg/l/h Control 4.0643.4 17.4 43 Y23/101-59 (PDC) 4.28 51.6 19.9 46 Y23/81/101-4 3.39 55.718.6 36 (PDC + ALD Y23/85/101-14 5.17 57.9 22.7 55 (PDC + ALD + ACS)Y23/86/101-23 4.00 53.4 21.1 43 (PDC + ACS) Y23/95/101-2 3.50 47.5 20.137 (PDC + PDAT) Y23/81/95/101-20 4.38 62.0 24.0 47 (PDC + ALD + PDAT)Y23/85/95/101-8 4.55 54.4 21.5 48 (PDC + ALD + ACS + PDAT)

TABLE 19B Triacylglycerol (TAG) concentration (g/l) in the culturemedium and calculated total TAG concentration (g/l), rate (mg/l/h) andyield (%) per used glucose in cultivation after 94 hours cultivation inglucose medium with C/N ratio of 153 Yield total TAG TAG Total TAG (%used total TAG Strain (g/l) (g/l) glucose) mg/l/h Control 0.04 4.10 17.644 Y23/101-59 (PDC) 0.01 4.29 20.0 46 Y23/81/101-4 0.25 3.63 19.9 39(PDC + ALD Y23/85/101-14 0.03 5.20 22.8 55 (PDC + ALD + ACS)Y23/86/101-23 0.18 4.18 22.1 44 (PDC + ACS) Y23/95/101-2 0.01 3.51 20.237 (PDC + PDAT) Y23/81/95/101-20 0.03 4.42 24.2 47 (PDC + ALD + PDAT)Y23/85/95/101-8 0.04 4.6 21.7 49 (PDC + ALD + ACS + PDAT)

This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations and ratesof production and triacylglycerol yields from used glucose or per dryweight in the yeast cell and/or in the culture medium with high C/Nratio in different stages of the cultivation.

Example 40. Aerobic Shake Flask Characterization of Strains Y23/101-55(Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-19 (Ex. 37B),Y23/86/101-23 (Ex. 37B), Y23/95/101-2 (Ex. 37B) and Y23/85/95/101-7 (Ex.37B) in Glucose Medium with C/N Ratio of 28

Transformants were separately cultivated in 50 ml of Glucose-CN28 medium(pH 5.5, 30 g glucose, 0.3 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5 gNa₂HPO₄*2H₂O, 1.5 g MgSO₄*7H₂O, 4.0 g Yeast extract, 51 mg CaCl₂, 8 mgFeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) wasinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusglucose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 250 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis were withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 was used as acontrol. Cell dry weight was determined and HPLC analysis was carriedout as described in Example 22. Lipid extraction from the yeast cellsand triacylglycerol measurements were carried out as described inExample 21 and lipid extraction from the culture medium as described inExample 39.

After 46 hours cultivation (Table 20A), when 12 to 14 g/l glucose wasleft, Y23/101-55, Y23/81/101-4, Y23/85/101-19, Y23/86/101-23,Y23/95/101-2 and Y23/85/95/101-7 transformants produced 18, 17, 20, 24,7 and 17% more triacylglycerol with higher rate, respectively, than thecontrol strain in glucose medium. Also triacylglycerol yields on biomassand per used glucose were 10 to 35% and 7 to 26% higher with thetransformants than the control strain, respectively. After 72 and 94hours cultivation triacylglycerol concentration in the culture mediumwith the transformants Y23/81/101-4 and Y23/86/101-23 was 195 and 56%and 35 and 30% higher, respectively, than in the cultivations with thecontrol strain (Table 20B). Additionally, yield of triacylglyceroldetected in culture medium per used glucose was 57 to 206% and 36 to 56%higher with the transformants Y23/81/101-4 and Y23/86/101-23 than withthe control after 72 and 94 hours cultivation, respectively.

TABLE 20A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) andyield (%) per biomass (CDW) and used glucose in the yeast cells after 46hours cultivation in glucose medium with C/N ratio of 28 Yield TAG TAGYield TAG (% used TAG Strain (g/l) (% CDW) glucose) mg/l/h Control 3.1046.6 20.6 67 Y23/101-55 (PDC) 3.67 51.2 24.4 80 Y23/81/101-4 3.64 53.522.1 79 (PDC + ALD Y23/85/101-19 3.71 53.8 23.2 81 (PDC + ALD + ACS)Y23/86/101-23 3.84 63.1 26.0 83 (PDC + ACS) Y23/95/101-2 3.31 54.7 23.372 (PDC + PDAT) Y23/85/95/101-7 3.64 58.5 26.3 79 (PDC + ALD + ACS +PDAT)

TABLE 20B Triacylglycerol (TAG) concentration (g/l) and yield (%) perused glucose in the culture medium after 72 and 94 hours cultivation inglucose medium with C/N ratio of 28 72 h Yield TAG % 94 h Yield TAG TAG(/used TAG (% used Strain (g/l) glucose) (g/l) glucose) Control 0.853.19 2.79 9.84 Y23/81/101-4 2.51 9.75 4.35 15.4 (PDC + ALD)Y23/86/101-23 1.15 5.00 3.62 13.4 (PDC + ACS)

This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations and ratesof production and triacylglycerol yields from used glucose or per dryweight in the yeast cell and/or in the culture medium with low C/N ratioat different stages of the cultivation.

Example 41. Aerobic Shake Flask Characterization of Strains Y23/101-57(Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-13 (Ex. 37B),Y23/86/101-23 (Ex. 37B), Y23/95/101-1 (Ex. 37B), Y23/81/95/101-20 (Ex.37B) and Y23/85/95/101-8 (Ex. 37B) in Xylose Medium with C/N Ratio of103

Transformants were separately cultivated in 50 ml of Yeast culturemedium IV (pH 5.5, 20 g xylose, 0.15 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5 gNa₂HPO₄*2 H₂O, 1.5 g MgSO₄*7H₂O, 0.45 g Yeast extract, 51 mg CaCl₂, 8 mgFeCl₃*6H₂O and 0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) wasinoculated to an OD₆₀₀ of 0.3 with cells grown on yeast peptone plusxylose plates. The cultivations were maintained at a temperature of 30°C. with shaking at 250 rpm. Samples for cell dry weight measurement,lipid extraction and HPLC analysis were withdrawn periodically duringcultivation. Cryptococcus curvatus wild type strain Y23 was used as acontrol. Cell dry weight was determined and HPLC analysis was carriedout as described in Example 22. Lipid extraction from the yeast cellsand triacylglycerol measurements were carried out as described inExample 21 and lipid extraction from the culture medium as described inExample 39.

After 47 hours cultivation (Table 21) Y23/101-57, Y23/81/101-4,Y23/85/101-13 and Y23/95/101-1 transformants produced 12, 18, 4 and 5%more triacylglycerol with higher rate, respectively, than the controlstrain in xylose medium. Also triacylglycerol yields on biomass and perused xylose were up to 28% and 16% higher with the transformantsY23/101-57, Y23/81/101-4, Y23/85/101-13, Y23/86/101-23 and Y23/95/101-1than the control strain, respectively. Also total lipid concentration,rate and yields on biomass and per used xylose were 8 to 29%, 8 to 29%,19 to 44% and 11 to 33% higher with the transformants than the controlstrain, respectively, after 47 hours cultivation in xylose medium.

TABLE 21 Triacylglycerol (TAG) and total lipid concentrations (g/l),rates (mg/l/h) and yields (%) per biomass (CDW) and used xylose in theyeast cells after 47 hours cultivation in xylose medium with C/N ratioof 103 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) xylose)mg/l/h Control 1.90 38.5 16.0 41 Y23/101-57 (PDC) 2.12 44.7 18.0 45Y23/81/101-4 2.24 49.2 18.6 48 (PDC + ALD Y23/85/101-13 1.98 44.3 18.142 (PDC + ALD + ACS) Y23/86/101-23 1.85 47.7 18.3 39 (PDC + ACS)Y23/95/101-1 2.00 38.5 16.5 43 (PDC + PDAT) Yield lipid Lipid Yieldlipid (% used Lipid Strain (g/l) (% CDW) xylose) mg/l/h Control 2.4048.5 20.2 51 Y23/101-57 (PDC) 2.75 57.9 23.3 59 Y23/81/101-4 2.70 59.322.4 57 (PDC + ALD Y23/85/101-13 2.60 58.1 23.7 55 (PDC + ALD + ACS)Y23/86/101-23 2.70 69.7 26.8 57 (PDC + ACS) Y23/95/101-1 3.10 59.6 25.666 (PDC + PDAT)

After 94 hours cultivation (Table 22A) Y23/101-57, Y23/81/101-4,Y23/85/101-13, Y23/86/101-23, Y23/95/101-1, Y23/81/95/101-20 andY23/85/95/101-8 transformants produced 7, 9, 8, 7, 6, 6 and 4% moretriacylglycerol with higher rate, respectively, than the control strainin xylose medium. Also triacylglycerol yields on biomass and per usedxylose were 4 to 25% and 5 to 17% higher with the transformants than thecontrol strain, respectively. Triacylglycerol concentration in theculture medium with the transformants Y23/81/101-4 and Y23/86/101-23 was222 and 156% higher, respectively, than in the cultivations with thecontrol strain (Table 22B). Additionally, the total triacylglycerolyields per used xylose calculated from the intracellular triacylglycerolconcentration and the triacylglycerol concentration detected from theculture medium were 7 to 17% higher with the transformants than with thecontrol strain.

TABLE 22A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) andyield (%) per biomass (CDW) and used xylose in the yeast cells after 94hours cultivation in xylose medium with C/N ratio of 103 Yield TAG TAGYield TAG (% used TAG Strain (g/l) (% CDW) xylose) mg/l/h Control 4.5072.0 23.9 48 Y23/101-57 (PDC) 4.82 79.4 25.6 51 Y23/81/101-4 4.92 84.226.1 52 (PDC + ALD Y23/85/101-13 4.85 75.1 25.7 52 (PDC + ALD + ACS)Y23/86/101-23 4.82 88.5 25.6 51 (PDC + ACS) Y23/95/101-1 4.79 79.8 25.751 (PDC + PDAT) Y23/81/95/101-20 4.78 89.7 27.9 51 (PDC + ALD + PDAT)Y23/85/95/101-8 4.66 80.4 25.2 50 (PDC + ALD + ACS + PDAT)

TABLE 22B Triacylglycerol (TAG) concentration (g/l) in the culturemedium and calculated total TAG concentration (g/l), rate (mg/l/h) andyield (%) per used xylose in cultivation after 94 hours cultivation inxylose medium with C/N ratio of 103 Yield total TAG TAG Total TAG (%used total TAG Strain (g/l) (g/l) xylose) mg/l/h Control 0.09 4.59 24.349 Y23/101-57 (PDC) 0.07 4.89 26.0 52 Y23/81/101-4 0.29 5.22 27.7 55(PDC + ALD Y23/85/101-13 0.10 4.94 26.2 53 (PDC + ALD + ACS)Y23/86/101-23 0.23 5.05 26.8 54 (PDC + ACS) Y23/95/101-1 0.07 4.86 26.152 (PDC + PDAT) Y23/81/95/101-20 0.09 4.86 28.4 52 (PDC + ALD + PDAT)Y23/85/95/101-8 0.10 4.76 25.7 51 (PDC + ALD + ACS + PDAT)

This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol and total lipidconcentrations and rates of production and triacylglycerol and totallipid yields from used xylose or per dry weight in the yeast cell and/orin the culture medium with high C/N ratio in different stages of thecultivation.

Example 42. Aerobic Shake Flask Characterization of Strains Y23/101-57(Ex. 37B), Y23/85/101-13 (Ex. 37B) and Y23/81/95/101-20 (Ex. 37B) inXylose Medium with C/N Ratio of 20

Transformants were separately cultivated in 50 ml of Xylose-CN20 medium(pH 5.5, 20 g xylose, 0.3 g (NH₄)₂SO₄, 7.0 g KH₂PO₄, 2.5 g Na₂HPO₄*2H₂O,1.5 g MgSO₄*7H₂O, 4.0 g Yeast extract, 51 mg CaCl₂, 8 mg FeCl₃*6H₂O and0.1 mg ZnSO₄*7H₂O per litre). Each flask (250 ml) was inoculated to anOD₆₀₀ of 0.3 with cells grown on yeast peptone plus xylose plates. Thecultivations were maintained at a temperature of 30° C. with shaking at250 rpm. Samples for cell dry weight measurement, lipid extraction andHPLC analysis were withdrawn periodically during cultivation.Cryptococcus curvatus wild type strain Y23 was used as a control. Celldry weight was determined and HPLC analysis was carried out as describedin Example 22. Lipid extraction from the yeast cells and triacylglycerolmeasurements were carried out as described in Example 21 and lipidextraction from the culture medium as described in Example 39.

After 94 hours cultivation (Table 23A) transformants Y23/101-57 andY23/85/101-13 produced 9 and 5% more triacylglycerol with higher rate,respectively, than the control strain in xylose medium. Alsotriacylglycerol yields on biomass and per used xylose were 2 to 10% and4 to 8% higher with the transformants than the control strain,respectively. Triacylglycerol concentration in the culture medium withthe transformants Y23/101-57, Y23/85/101-13 and Y23/81/95/101-20 was163, 163 and 188% higher, respectively, than in the cultivations withthe control strain (Table 21B). Additionally, the total triacylglycerolyields per used xylose calculated from the intracellular triacylglycerolconcentration and the triacylglycerol concentration detected from theculture medium were 4 to 12% higher with the transformants than with thecontrol strain.

TABLE 23A Triacylglycerol (TAG) concentration (g/l) and yield (%) perbiomass (CDW) and used xylose in the yeast cells after 94 hourscultivation in xylose medium with C/N ratio of 20 Yield TAG TAG YieldTAG (% used Strain (g/l) (% CDW) xylose) Control 4.21 68.7 21.1Y23/101-57 (PDC) 4.57 75.8 22.8 Y23/85/101-13 4.41 69.9 22.0 (PDC +ALD + ACS)

TABLE 23B Triacylglycerol (TAG) concentration (g/l) in the culturemedium and calculated total TAG concentration (g/l), rate (mg/l/h) andyield (%) per used xylose in cultivation after 94 hours cultivation inxylose medium with C/N ratio of 20 Yield total TAG TAG Total TAG (% usedtotal TAG Strain (g/l) (g/l) xylose) mg/l/h Control 0.08 4.28 21.4 46Y23/101-57 (PDC) 0.21 4.78 23.9 51 Y23/85/101-13 0.21 4.61 23.1 49(PDC + ALD + ACS) Y23/81/95/101-20 0.23 4.45 22.3 47 (PDC + ALD + PDAT)

This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes indifferent combinations enhanced triacylglycerol concentrations and ratesof production and triacylglycerol yields from used xylose or per dryweight in the yeast cell and/or in the culture medium with low C/Nratio.

Example 43. Aerobic Shake Flask Characterization of Strains M22/94-16(Ex. 16B) and M22/94/102-31 (Ex. 38B), in Xylose Medium with C/N Ratioof 21

Transformants were separately cultivated in 50 ml of mould C/N 21 medium(pH 5.5, 10 g glucose, 1.4 g yeast extract, 2.5 g KH₂PO₄, 0.3 g(NH₄)₂SO₄, 10 mg ZnSO₄*7H₂O, 2 mg CuSO₄*5H₂O, 10 mg MnSO₄, 0.5 gMgSO₄*7H₂O, 0.1 g CaCl₂, 20 mg FeCl₃*6H₂O per litre). Each flask (250ml) was inoculated with 1*10⁷ spores. The cultivations were maintainedat a temperature of 28° C. with shaking at 250 rpm. Samples for cell dryweight measurement, lipid extraction and HPLC analysis were withdrawnperiodically during cultivation. Mucor circinelloides wild type strainM22 was used as a control. Cell dry weight was determined as describedin Example 25 and HPLC analysis was carried out as described in Example22. Lipid extraction and total lipid and triacylglycerol measurements asdescribed in Example 21.

After 96 hours cultivation (Table 24) transformants M22/94-16 andM22/94/102-31 produced 18 and 30% more triacylglycerol with higher rate,respectively, than the control strain in xylose medium. Alsotriacylglycerol yields on biomass and per used xylose were 10 to 34% and1 to 24% higher with the transformants than the control strain,respectively. Also total lipid concentration and yield on biomass were15 to 16% and 6 to 33% higher in the transformants than in the control.

TABLE 24 Triacylglycerol (TAG) and total lipid concentrations (g/l),rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) andused xylose after 96 hours cultivation in xylose medium with C/N ratioof 21 Yield TAG TAG Yield TAG (% used TAG Strain g/l (% CDW) xylosemg/l/h Control 0.60 34.9 13.9 6 M22/94-16 0.71 38.4 14.0 7 (ALD + ACS)M22/94/102-31 0.78 53.2 17.2 8 (PDC + ALD + ACS) Yield lipid Lipid Yieldlipid (% used Lipid Strain g/l (% CDW) xylose mg/l/h Control 0.81 47.618.9 8 M22/94-16 0.94 50.5 18.5 10 (ALD + ACS) M22/94/102-31 0.93 63.520.5 10 (PDC + ALD + ACS)

This example shows that expression of PDC1, ALD6 and ACS2 genes indifferent combinations enhanced triacylglycerol and total lipidconcentrations and rates of production and triacylglycerol and totallipid yields from used xylose or per dry weight in the yeast cellculture me with low C/N ratio.

Example 44. Production of Triacylglycerol or Lipid by Strains of C.curvatus Modified by Addition of Genes Encoding PDC and ALD and PDAT andACS (Y23/85/95/101-8, Ex. 37B) in High Cell Density Cultures Grown onGlucose with C/N Ratio of 28

Transformant (Y23/85/95/101-8) was cultivated in Multifors bioreactors(max. working volume 500 ml, Infors HT, Switzerland) at pH 4.0, 30° C.,in 500 ml medium containing 90 to 96 g glucose, 6.74 g (NH₄)₂SO₄, 1.2 gKH₂PO₄, 0.3 g Na₂HPO₄.2H₂O, 1.5 g MgSO₄.7H₂O, 0.1 g CaCl₂.6H₂O, 5.26 mgcitric acid.H₂O, 5.26 mg ZnSO₄.7H₂O, 0.1 mg MnSO₄.4H₂O, 0.5 mgCoCl₂.6H₂O, 0.26 mg CuSO₄.5H₂O, 0.1 mg Na₂MoO₄.2H₂O, 1.4 mg FeSO₄.7H₂O,0.1 mg H₃BO₄, 0.05 mg D-biotin, 1.0 mg CaPantothenate, 5.0 mg nicotinicacid, 25 mg myoinositol, 1.0 mg thiamine.HCl, 1.0 mg pyridoxine.HCl and0.2 mg p-aminobenzoic acid per litre. The pH was maintained constant byaddition of 1 M KOH or 1 M H₂PO₄. Cultures were agitated at 1000 rpm (2Rushton turbine impellors) and aerated at 2 volumes air per volumeculture per minute (vvm). Clerol FBA 3107 antifoaming agent (Cognis,SaintFargeau-Ponthierry Cedex France, 1 ml l⁻¹) was added to preventfoam accumulation. Bioreactors were inoculated to initial OD₆₀₀ of 0.5to 4.0 with cells grown in the same medium (substituting 1.5 g urea perlitre for (NH₄)₂SO₄ and omitting the CaCl₂.6H₂O) in 50 ml volumes in 250ml flasks at 30° C. with shaking at 200 rpm for 24 to 42 h. Samples forcell dry weight measurement, lipid extraction and HPLC analysis werewithdrawn periodically during cultivation. Cryptococcus curvatus wildtype strain Y23 was used as the control.

Lipid extraction and triacylglycerol concentration measurements werecarried out as described in Example 21. Lipid extraction from theculture medium was carried out as described in Example 39. Cell dryweight was determined as described in Example 30. HPLC analyses werecarried out as described in Example 22.

Table 25A shows that a transformant containing the genes PDC, ALD, ACSand PDAT produced 58% more triacylglycerol than Y23, with a 67% and 185%increase in the yields on glucose consumed and on biomass, respectively.Additionally, the transformant containing the genes PDC, ALD, ACS andPDAT produced 20% more triacylglycerol than Y23, with a 111% increase inthe yield on glucose consumed in the culture medium (Table 25B).

TABLE 25A Triacylglycerol produced in pH controlled bioreactor cultureof Y23 and transformant of Y23 expressing PDC + ALD + ACS + PDAT, withglucose as carbon source and C/N 28. Data is the average of 2 cultures ±standard error of the mean. Percentage increase is shown in parenthesis.Yield TAG TAG (% glucose Yield TAG Strain (g/l) consumed) (% per CDW)Y23 1.30 ± 0.06 1.22 ± 0.01 2.67 ± 0.14 Y23/85/95/101-8 2.05 ± 0.34 2.04± 0.22 4.95 ± 0.33 (PDC + ALD + ACS + (+58%) (+67%) (+185%) PDAT)

TABLE 25B Triacylglycerol produced in the culture medium in pHcontrolled bioreactor culture of Y23 and transformant of Y23 expressingPDC + ALD + ACS + PDAT, with glucose as carbon source and C/N 28. Datais the average of 2 cultures ± standard error of the mean. Percentageincrease is shown in parenthesis. Yield TAG TAG (% glucose Strain (g/l)consumed) Y23 0.15 ± 0.04 0.18 ± 0.03 Y23/85/95/101-8 0.18 ± 0.00 0.38 ±0.07 (PDC + ALD + ACS + (+20%) (+111%) PDAT)

This example shows that expression of PDC1, ALD6, ACS1 and PDAT enhancedtriacylglycerol concentrations and triacylglycerol yields from usedglucose or per dry weight in high cell density cultures in the yeastcell and/or in the culture medium.

Sequences used:

SEQ ID NOs: 1 and 2 correspond to primers YeastTEF1 and YeastTEF4,respectively, used to isolate genomic fragment of the C. curvatus TEFgene.

SEQ ID NOs: 3 and 4 correspond to primers PCR linker I and PCR linkerII, respectively, used in chromosome walk experiments.

SEQ ID NOs: 5, 6, 7 and 8 correspond to primers CC_TEF2, CC_TEF1,CC_TEF6 and CC_TEF5 respectively, used to isolate genomic fragments ofthe C. curvatus TEF promoter region in chromosome walk experiments.

SEQ ID NOs: 9 and 10 correspond to primers CC_TEF10 and CC_TEF11,respectively, used to isolate promoter of the C. curvatus TEF gene.

SEQ ID NOs: 11 and 12 correspond to primers CC_TEF3 and CC_TEF4,respectively, used to isolate genomic fragment of the C. curvatus TEFterminator region in chromosome walk experiments.

SEQ ID NOs: 13 and 14 correspond to primers CC_TEF7 and CC_TEF8,respectively, used to isolate terminator of the C. curvatus TEF gene.

SEQ ID NOs: 15 and 16 correspond to primers Yeast TPI5 and Yeast TPI8,respectively, used to isolate genomic fragment of the C. curvatus TPIgene.

SEQ ID NOs: 17 and 18 correspond to primers CC_TPI2 and CC_TPI1,respectively, used to isolate genomic fragment of the C. curvatus TPIpromoter region in chromosome walk experiments.

SEQ ID NOs: 19 and 20 correspond to primers CC_TPI7 and CC_TPI9,respectively, used to isolate promoter of the C. curvatus TPI gene.

SEQ ID NOs: 21 and 22 correspond to primers CC_TPI4 and CC_TPI3,respectively, used to isolate genomic fragment of the C. curvatus TPIterminator region in chromosome walk experiments.

SEQ ID NOs: 23 and 24 correspond to primers CC_TPI5 and CC_TPI6,respectively, used to isolate terminator of the C. curvatus TPI gene.

SEQ ID NOs: 25 and 26 correspond to primers YeastENO5 and YeastENO10,respectively, used to isolate genomic fragment of the C. curvatus ENOgene.

SEQ ID NOs: 27, 28, 29 and 30 correspond to primers CC_ENO2, CC_ENO1,CC_ENO5 and CC_ENO6, respectively, used to isolate genomic fragments ofthe C. curvatus ENO promoter region in chromosome walk experiments.

SEQ ID NOs: 31 and 32 correspond to primers CC_ENO9 and CC_ENO10,respectively, used to isolate promoter of the C. curvatus ENO gene.

SEQ ID NOs: 33 and 34 correspond to primers CC_ENO4 and CC_ENO3,respectively, used to isolate genomic fragment of the C. curvatus ENOterminator region in chromosome walk experiments.

SEQ ID NOs: 35 and 36 correspond to primers CC_ENO7 and CC_ENO8,respectively, used to isolate terminator of the C. curvatus ENO gene.

SEQ ID NOs: 37 and 38 correspond to primers CC_GPD3 and CC_GPD4,respectively, used to isolate genomic fragment of the C. curvatus GPDterminator region in chromosome walk experiments.

SEQ ID NOs: 39 and 40 correspond to primers CC_GPD6 and CC_GPD7,respectively, used to isolate terminator of the C. curvatus GPD gene.

SEQ ID NOs: 41 and 42 correspond to primers Hph 5 and Hph 3,respectively, used to isolate E. coli hygromycin gene.

SEQ ID NOs: 43 and 44 correspond to primers Kan 5 and Kan 3,respectively, used to isolate E. coli G418 resistance gene.

SEQ ID NOs: 45 and 46 correspond to primers CERR 5 and CERR 3,respectively, used to isolate S. cerevisiae cerulenin resistance gene.

SEQ ID NO: 47 corresponds to the amino acid sequence of the S.cerevisiae ALD6 gene, with GenBank accession number AAB68304 (versionnumber AAB68304.1).

SEQ ID NO: 48 corresponds to S. cerevisiae ALD6 protein encoding DNAcodon optimized according to Ustilago maydis-fungus codon usage.

SEQ ID NO: 49 corresponds to S. cerevisiae ALD6 protein encoding DNAcodon optimized according to Rhizopus oryzae-filamentous fungus codonusage.

SEQ ID NO: 50 corresponds to the amino acid sequence of the S.cerevisiae ACS2 gene, with GenBank accession number CAA97725 (versionnumber CAA97725.1.

SEQ ID NO: 51 corresponds to S. cerevisiae ACS2 protein encoding DNAcodon optimized according to Ustilago maydis-fungus codon usage.

SEQ ID NO: 52 corresponds to the amino acid sequence of the Rhizopusoryzae PDAT gene, encoded by gene with locus number RO3G_07851.3 inBroad Institute Rhizopus oryzae database.

SEQ ID NO: 53 corresponds to Rhizopus oryzae PDAT protein encoding DNAcodon optimized according to Ustilago maydis-fungus codon usage.

SEQ ID NOs: 54 and 55 correspond to primers Mould TPI1 and mould TPI3,respectively, used to isolate genomic fragment of the Mucorcircinelloides TPI gene.

SEQ ID NOs: 56 and 57 correspond to primers MC_TPI2 and MC_TPI1,respectively, used to isolate genomic fragment of the Mucorcircinelloides TPI promoter region in chromosome walk experiments.

SEQ ID NOs: 58 and 59 correspond to primers MC_TPI7 and MC_TPI8,respectively, used to clone promoter of the Mucor circinelloides TPIgene.

SEQ ID NOs: 60 and 61 correspond to primers MC_TPI4 and MC_TPI3,respectively, used to isolate genomic fragment of the Mucorcircinelloides TPI terminator region in chromosome walk experiments.

SEQ ID NOs: 62 and 63 correspond to primers MC_TPI5 and MC_TPI6,respectively, used to clone terminator of the Mucor circinelloides TPIgene.

SEQ ID NOs: 64 and 65 correspond to primers Mould TEF1 and Mould TEF4,respectively, used to isolate genomic fragment of the Mucorcircinelloides TEF gene.

SEQ ID NOs: 66, 67, 68 and 69 correspond to primers MC_TEF2, MC_TEF1,MC_TEF6 and MC_TEF5, respectively, used to isolate genomic fragments ofthe Mucor circinelloides TEF promoter region in chromosome walkexperiments.

SEQ ID NOs: 70 and 71 correspond to primers MC_TEF9 and MC_TEF10,respectively, used to clone promoter of the Mucor circinelloides TEFgene.

SEQ ID NOs: 72, 73, 74 and 75 correspond to primers MC_TEF4, MC_TEF3,MC_TEF8 and MC_TEF7, respectively, used to isolate genomic fragments ofthe Mucor circinelloides TEF terminator region in chromosome walkexperiments.

SEQ ID NOs: 76 and 77 correspond to primers MC_TEF11 and MC_TEF12,respectively, used to clone terminator of the Mucor circinelloides TEFgene.

SEQ ID NOs: 78 and 79 correspond to primers Mould PGK4 and Mould PGK2,respectively, used to isolate genomic fragment of the Mucorcircinelloides PGK gene.

SEQ ID NOs: 80, 81, 82 and 83 correspond to primers MC_PGK2, MC_PGK1,MC_PGK4 and MC_PGK3, respectively, used to isolate genomic fragments ofthe Mucor circinelloides PGK promoter region in chromosome walkexperiments.

SEQ ID NOs: 84 and 85 correspond to primers MC_PGK5 and MC_PGK6,respectively, used to clone promoter of the Mucor circinelloides PGKgene.

SEQ ID NOs: 86, 87, 88 and 89 correspond to primers MC_GPD2, MC_GPD1,MC_GPD10 and MC_GPD9, respectively, used to isolate genomic fragment ofthe Mucor circinelloides GPD promoter region in chromosome walkexperiments.

SEQ ID NOs: 90 and 91 correspond to primers MC_GPD11 and MC_GPD12,respectively, used to clone promoter of the Mucor circinelloides GPDgene.

SEQ ID NO: 92 corresponds to S. cerevisiae ACS2 protein encoding DNAcodon optimized according to Rhizopus oryzae-fungus codon usage.

SEQ ID NO: 93 corresponds to Rhizopus oryzae PDAT protein encoding DNAcodon optimised according to Rhizopus oryzae-fungus codon usage.

SEQ ID NO: 94 corresponds to S. cerevisiae PDC1 protein encoding DNA,with GenBank accession number X77316 (version number X77316.1).

SEQ ID NO: 95 corresponds to the amino acid sequence of the S.cerevisiae PDC1 gene, with GenBank accession number CAA54522 (versionnumber CAA54522.1.

SEQ ID NO: 96 corresponds to S. cerevisiae PDC1 protein encoding DNAcodon optimized according to Ustilago maydis-fungus codon usage.

SEQ ID NO: 97 corresponds to S. cerevisiae PDC1 protein encoding DNAcodon optimized according to Rhizopus oryzae-fungus codon usage.

REFERENCES

-   Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang,    Z., Miller, W. and Lipman, D. J. 1997. Nucleic Acids Res.    25:3389-3402.-   Boulton, C. A. and Ratledge, C. 1981. J. Gen. Microbiol.    127:169-176.-   Folch J., Lees M. and Stqanley, G. H. S. J. Biol. Chem 226:497-509    (1957)-   van den Berg, M. A., de Jong-Gubbels, P., Steensma, H. Y., van    Dijken, J. P. and Pronk, J. T. 1996. J. Biol. Chem. 271:28953-28959.-   Connerton, I. F., Fincham, J. R. S., Sandeman, R. A. and    Hynes, M. J. 1990. Mol. Microbiol. 4:451-460.-   Dahlqvist, A., Stahl, U., Lenman, M., Banas, A., Lee, M., Sandager,    L., Ronne, H. and Stymne, S. 2000. PNAS 97:6487-6492-   Flikweert, M. T., Van der Zanden, L., Janssen, W. M. TH. M.,    Steensma, H. Y., Van Dijken, J. P. and Pronk, J. T. 1996. Yeast    12:247-257.-   Flipphi, M., Mathieu, M., Cirpus, I., Panozzo, C. and    Felenbok, B. 2001. J. Biol. Chem. 276:6950-6958.-   Hiesinger, M., Wagner, C. and Schuller, H.-J. 1997. FEBS Lett.    415:16-20. Hynes, M. J. and Murray, S. L. 2010. Euk. Cell    9:1039-1048.-   Mach, R. L., Schindler, M. and Kubicek, C. P. 1994. Curr Genet. 25,    567-570 Meesters, P. A. E. P., Springer, J. and Eggink, G. 1997.    Appl. Microbiol. Biotechnol. 47:663-667.-   Mueller, P. R. and Wold, B. 1989, “In vivo footprinting of a muscle    specific enhancer by ligation mediated PCR.” Science 246:780-786.-   Nakazawa, N., Hashimoto, H., Harashima, S. and Oshima, Y. 1993. J.    Ferm. Bioeng. 76:60-63.-   Postma, E., Verduyn, C., Scheffers, W. A. and van    Dijken, J. P. 1989. Appl. Environ. Microbiol. 55:468-477.-   Pronk, J. T., Steensma, H. Y. and Van Dijken, J. P. 1996. Yeast    12:1607-1633.-   Ratledge, C. and Wynn, J. P. 2002. Adv. Appl. Microbiol. 51:1-51.-   Saint-Prix, F., Bonquist, L. and Dequin, S. 2004. Microbiol.    150:2209-2220.-   Sambrook, J. and Russell, D. W. 2001. Molecular cloning, a    laboratory manual. Cold Spring Harbor Laboratory, New York, US.-   Shiba, Y, Paradise, E M, Kirby J, Ro, D-K and Keasling J D. 2007.    Metabolic Engineering 9; 160-168.-   Skory, C. D. 2003. Curr. Microbiol. 47:59-64.-   Sorger D. and Daum G. (2003) Appl. Microbiol. Biotechnol 61:289-299.-   Takahashi, H., McCaffery, J. M., Irizarry, R. A. and    Boeke, J. D. 2006. Mol. Cell 23:207-217.-   Tehlivets, O., Scheuringer K. and Kohlwein S. D. (1997) Biocim    Biophys Acta 1771:255-270.-   Wolff, A. M., Appel, K. F., Petersen, J. B., Poulsen, U. and    Arnau, J. 2002 FEMS Yeast Res. 2:203-213

1. A genetically modified oleaginous fungal cell comprising: a) anucleic acid with enhanced expression encoding an acetaldehydedehydrogenase (ALD), and b) a nucleic acid with enhanced expressionencoding a diacylglycerol acyltransferase (DAT), wherein saidgenetically modified oleaginous fungal cell has enhanced lipidproduction in an aerobic bioreactor compared to a genetically unmodifiedoleaginous fungal cell.
 2. The genetically modified oleaginous fungalcell of claim 1, wherein the encoded ALD is a cytosolic ALD.
 3. Thegenetically modified oleaginous fungal cell of claim 2, wherein theencoded ALD is a fungal ALD, preferably Saccharomyces cerevisiae ALD6.4. The genetically modified oleaginous fungal cell of claim 1, whereinthe DAT encoding nucleic acid encodes a phosholipid:diacylglycerolacyltransferase (PDAT).
 5. The genetically modified oleaginous fungalcell of claim 4, wherein the encoded DAT is a fungal DAT, preferably ofRhizopus oryzae, and most preferably it encodes aphosholipid:diacylglycerol acyltransferase (PDAT) having at least 40%sequence identity to SEQ ID NO:52, or an enzymatically active fragmentor variant thereof.
 6. The genetically modified oleaginous fungal cellof claim 1, further comprising a nucleic acid with enhanced expressionencoding acetyl-CoA synthetase (ACS).
 7. The genetically modifiedoleaginous fungal cell of claim 6, wherein the nucleic acid encoding ACSis a gene that is not under glucose repression or its gene product isnot subject to post-translational regulation.
 8. The geneticallymodified oleaginous fungal cell of claim 7, wherein the encoded ACS is afungal ACS, preferably Saccharomyces cerevisiae ACS2.
 9. The geneticallymodified oleaginous fungal cell of claim 1, which is a yeast cellselected from the genera Cryptococcus, Candida, Galactomyces, Hansenula,Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia,preferably from the group consisting of Candida sp., Cryptococcuscurvatus, Cryptococcus albidus, Galactomyces geotrichum, Hansenulaciferri, Lipomyces lipofer, Lipomyces ssp., Lipomyces starkeyi,Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis,Trichosporon pullulans and Yarrowia lipolytica, or a filamentous fungalcell selected from the genera Aspergillus, Cunninghamella, Fusarium,Glomus, Humicola, Mortierella, Mucor, Penicillium, Pythium and Rhizopus,preferably from the group consisting of Aspergillus nidulans,Aspergillus oryzae, Aspergillus terreus, Aspergillus niger,Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius, Humicolalanuginose, Mortierella isabellina, Mortierella pusilla, Mortierellavinacea, Mucor circinelloides, Mucor plumbeus, Mucor ramanniana,Penicillium lilacinum, Penicillium spinulosum, Pythium ultimum andRhizopus oryzae.
 10. The genetically modified oleaginous fungal cell ofclaim 9, which is from the genera Cryptococcus or Mucor, preferably itis a cell of Cryptococcus curvatus or Mucor circinelloides.
 11. A methodof producing lipids, comprising cultivating a genetically modifiedoleaginous fungal cell according to claim 1 in a medium containingcarbon and nitrogen sources, and recovering the lipids produced.
 12. Themethod of claim 11, wherein the lipids are recovered from the culturemedium.
 13. The method of claim 11, wherein lipids comprisingacylglycerols, preferably triacylglycerols (TAG) are produced.
 14. Themethod of claim 11, wherein the carbon source is a hexose or pentosesugars containing material.
 15. The method of claim 11, comprisingproducing precursors for functional fatty acids.
 16. A method ofproducing biofuel, or lubricant, said method comprising cultivating agenetically modified oleaginous fungal cell according to claim 1 in amedium containing carbon and nitrogen sources, and recovering the lipidsproduced, and optionally esterifying said lipids to obtain biodiesel orlubricant, or hydrogenizing the lipids to obtain renewable diesel orlubricant.
 17. A method of preparing an oleaginous fungal cell of claim1, said method comprising transforming a fungal cell with a) a nucleicacid with enhanced expression encoding an acetaldehyde dehydrogenase(ALD) enzyme, and b) a nucleic acid with enhanced expression encoding adiacylglycerol acyltransferase (DAT).
 18. Use of a genetically modifiedfungal cell of claim 1 for producing lipids, precursors of functionalfatty acids, functional fatty acids, biofuels, biodiesel, renewablediesel or lubricants.